β-secretase enzyme compositions and methods

ABSTRACT

Disclosed are various forms of an active, isolated β-secretase enzyme in purified and recombinant form. This enzyme is implicated in the production of amyloid plaque components which accumulate in the brains of individuals afflicted with Alzheimer&#39;s disease Recombinant cells that produce this enzyme either alone or in combination with some of its natural substrates (β-APPwt and β-APPsw) are also disclosed, as are antibodies directed to such proteins. These compositions are useful for use in methods of selecting compounds that modulate β-secretase. Inhibitors of β-secretase are implicated as therapeutics in the treatment of neurodegenerative diseases, such as Alzheimer&#39;s disease.

This application is a concinuation of U.S. patent application Ser. No.09/501,708 filed Jan. 10, 2000, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 09/471,669filed Dec. 24, 1999, both of which claim the benefit of U.S. provisionalapplication Nos. 60/119,571 filed Feb. 10, 1999 and 60/139,172 filedJun. 15, 1999. 09/471,669 also claims priority to 60/114,408, filed Dec.31, 1998. U.S. patent application Ser. Nos. 09/501,708 and 60/119,571and 60/139,172 are hereby incorporated herein by reference in theirentierties.

FIELD OF THE INVENTION

The invention relates to the discovery of various active forms ofβ-secretase, an enzyme that cleaves β-amyloid precursor protein (APP) atone of the two cleavage sites necessary to produce β-amyloid peptide(Aβ). The invention also relates to inhibitors of this enzyme, which areconsidered candidates for therapeutics in the treatment of amyloidogenicdiseases such as Alzheimer's disease. Further aspects of the presentinvention include screening methods, assays, and kits for discoveringsuch therapeutic inhibitors, as well as diagnostic methods fordetermining whether an individual carries a mutant form of the enzyme.

BACKGROUND OF THE INVENTION

Alzheimer's disease is characterized by the presence of numerous amyloidplaques and neurofibrillatory tangles present in the brain, particularlyin those regions of the brain involved in memory and cognition.β-amyloid peptide (Aβ) is a 39–43 amino acid peptide that is majorcomponent of amyloid plaques and is produced by cleavage of a largeprotein known as the amyloid precursor protein (APP) at a specificsite(s) within the N-terminal region of the protein. Normal processingof APP involves cleavage of the protein at point 16–17 amino acidsC-terminal to the N-terminus of the β-AP region, releasing a secretedectodomain, α-sAPP, thus precluding production of β-AP. Cleavage byβ-secretase enzyme of APP between Met⁶⁷¹ and Asp⁶⁷² and subsequentprocessing at the C-terminal end of APP produces Aβ peptide, which ishighly implicated in the etiology of Alzheimer's pathology (Seubert, etal., in Pharmacological Treatment of Alzheimer's disease, Wiley-Liss,Inc., pp. 345–366, 1997; Zhao, J., et al. J. Biol. Chem. 271:31407–31411, 1996).

It is not clear whether β-secretase enzyme levels and/or activity isinherently higher than normal in Alzheimer's patients; however, it isclear that its cleavage product, Aβ peptide, is abnormally concentratedin amyloid plaques present in their brains. Therefore, it would bedesirable to isolate, purify and characterize the enzyme responsible forthe pathogenic cleavage of APP in order to help answer this and otherquestions surrounding the etiology of the disease. In particular, it isalso desirable to utilize the isolated enzyme, or active fragmentsthereof, in methods for screening candidate drugs for ability to inhibitthe activity of β-secretase. Drugs exhibiting inhibitory effects onβ-secretase activity are expected to be useful therapeutics in thetreatment of Alzheimer's disease and other amyloidogenic disorderscharacterized by deposition of Aβ peptide containing fibrils.

U.S. Pat. No. 5,744,346 (Chrysler, et al.) describes the initialisolation and partial purification of β-secretase enzyme characterizedby its size (apparent molecular weight in the range of 260 to 300kilodaltons when measured by gel exclusion chromatography) and enzymaticactivity (ability to cleave the 695-amino acid isotype of β-amyloidprecursor protein between amino acids 596 and 597). The presentinvention provides a significant improvement in the purity ofβ-secretase enzyme, by providing a purified β-secretase enzyme that isat least 200 fold purer than that previously described. Such a purifiedprotein has utility in a number of applications, includingcrystallization for structure determination. The invention also providesmethods for producing recombinant forms of β-secretase enzymes that havethe same size and enzymatic profiles as the naturally occurring forms.It is a further discovery of the present invention that humanβ-secretase is a so-called “aspartyl” (or “aspartic”) protease.

SUMMARY OF THE INVENTION

This invention is directed to a β-secretase protein that has now beenpurified to apparent homogeneity, and in particular to a purifiedprotein characterized by a specific activity of at least about 0.2×10⁵and preferably at least 1.0×10⁵ nM/h/μg protein in a representativeβ-secretase assay, the MBP-C 125sw substrate assay. The resultingenzyme, which has a characteristic activity in cleaving the 695-aminoacid isotype of β-amyloid precursor protein (β-APP) between amino acids596 and 597 thereof, is at least 10,000-fold, preferably at least20,000-fold and, more preferably in excess of 200,000-fold higherspecific activigy than an activity exhibited by a solubilized butunenriched membrane fraction from human 293 cells, such as have beenearlier characterized.

In one embodiment, the purified enzyme is fewer than 450 amino acids inlength, comprising a polypeptide having the amino acid sequence SEQ IDNO: 70 [63–452]. In preferred embodiments, the purified protein existsin a variety of “truncated forms” relative to the proenzyme referred toherein as SEQ ID NO: 2 [1–501], such as forms having amino acidsequences SEQ ID NO: 70 [63–452], SEQ ID NO: 69 [63–501], SEQ ID NO: 67[58–501], SEQ ID NO: 68 [58–452], SEQ ID NO: 58 [46–452], SEQ ID NO: 74[22–452]. More generally, it has been found that particularly usefulforms of the enzyme, particularly with regard to the crystallizationstudies described herein, are characterized by an N-terminus at position46 with respect to SEQ ID NO: 2 and a C-terminus between positions 452and 470 with respect to SEQ ID NO: 2. and more particularly, by anN-terminus at position 22 with respect to SEQ ID NO: 2 and a C-terminusbetween positions 452 and 470 with respect to SEQ ID NO: 2. These formsare considered to be cleaved in the transmembrane “anchor” domain. Otherparticularly useful purified forms of the enzyme include: SEQ ID NO:43[46–501], SEQ ID NO: 66 [22–501], and SEQ ID NO: 2 [1–501]. Moregenerally, it is appreciated that useful forms of the enzyme have anN-terminal residue corresponding to a residue selected from the groupconsisting of residues 22,46,58 and 63 with respect to SEQ ID NO: 2 anda C-terminus selected from a residue between positions 452 and 501 withrespect to SEQ ID NO: 2 or a C-terminus between residue positions 452and 470 with respect to SEQ ID NO: 2. Also described herein are forms ofenzyme isolated from a mouse, exemplified by SEQ ID NO: 65.

This invention is further directed to a crystalline protein compositionformed from a purified β-secretase protein, such as the various proteincompositions described above. According to one embodiment, the purifiedprotein is characterized by an ability to bind to the βsecretaseinhibitor substrate P10-P4′sta D→V which is at least equal to an abilityexhibited by a protein having the amino acid sequence SEQ ID NO:70 SEQID NO: 71 [46–419], when the proteins are tested for binding to saidsubstrate under the same conditions. According to another embodiment,the purified protein forming the crystallization composition ischaracterized by a binding affinity for the β-secretase inhibitorsubstrate SEQ ID NO: 72 (P10—P4′sta D→V) which is at least 1/100 of anaffinity exhibited by a protein having the amino acid sequence SEQ IDNO: 43 [46–501], when said proteins are tested for binding to saidsubstrate under the same conditions. Proteins forming the crystallinecomposition may be glycosylated or deglycosylated.

The invention also includes a crystalline protein composition containinga β-secretase substrate or inhibitor molecule, examples of which areprovided herein, particularly exemplified by peptide-derived inhibitorssuch as SEQ ID NO: 78, SEQ ID NO: 72, SEQ ID NO: 81, and derivativesthereof. Generally useful inhibitors in this regard will have a K_(i) ofno more than about 50 μM to 0.5 mM.

Another aspect of the invention is directed to an isolated protein,comprising a polypeptide that (i) is fewer than about 450 amino acidresidues in length, (ii) includes an amino acid sequence that is atleast 90% identical to SEQ ID NO: 75 [63–423] including conservativesubstitutions thereof, and (iii) exhibits β-secretase activity, asevidenced by an ability to cleave a substrate selected from the groupconsisting of the 695 amino acid isotype of beta amyloid precursorprotein (β3APP) between amino acids 596 and 597 thereof, MBP-C125 wt andMBP-C125sw. Peptides which fit these criteria include, but are notlimited to polypeptides which include the sequence SEQ ID NO: 75[63–423], such as SEQ ID NO: 58 [46–452], SEQ ID NO: 58 [46–452], SEQ IDNO: 58 [46–452], SEQ ID NO: 74 [22–452], and may also includeconservative substitutions within such sequences.

According to a further embodiment, the invention includes isolatedprotein compositions, such as those described above, in combination witha β-secretase substrate or inhibitor molecule, such as MBP-C125 wt,MBP-C125sw, APP, APPsw, and β-secretase-cleavable fragments thereof.Additional β-secretase-cleavable fragments useful in this regard aredescribed in the specification hereof. Particularly useful inhibitorsinclude peptides derived from or including SEQ ID NO: 78, SEQ ID NO: 81and SEQ ID NO: 72. Generally, such inhibitors will have K₁s of less thanabout 1 μM. Such inhibitors may be labeled with a detectable reportermolecule. Such labeled molecules are particularly useful, for example,in ligand binding assays.

In accordance with a further aspect, the invention includes proteincompositions, such as those described above, expressed by a heterologouscell. In accordance with a further embodiment, such cells may alsoco-express a β-secretase substrate or inhibitor protein or peptide. Oneor both of the expressed molecules may be heterologous to the cell.

In a related embodiment, the invention includes antibodies that bindspecifically to a β-secretase protein comprising a polypeptide thatincludes an amino acid sequence that is at least 90% identical to SEQ IDNO: 75 [63–423] including conservative substitutions thereof, but whichlacks significant immunoreactivity with a protein a sequence selectedfrom the group consisting of SEQ ID NO: 2 [1–501] and SEQ ID NO: 43[46–501].

In a further related embodiment, the invention includes isolated nucleicacids comprising a sequence of nucleotides that encodes a β-secretaseprotein that is at least 95% identical to a protein selected from thegroup consisting of ID NO: 66 [22–501], SEQ ID NO: 43[46–501], SEQ IDNO: 57 [1–419], SEQ ID NO: 74 [22–452], SEQ ID NO: 58 [46–452], SEQ IDNO: 59 [1–452], SEQ ID NO: 60 [1–420], SEQ ID NO: 67 [58–501], SEQ IDNO: 68 [58–452], SEQ ID NO: 69 [63–501], SEQ ID NO: 70 [63–452], SEQ IDNO: 75 [63–423], and SEQ ID NO: 71 [46–419], or a complementary sequenceof any of such nucleotides. Specifically excluded from this nucleotideis a nucleic acid encoding a protein having the sequence SEQ ID NO: 2[1–501].

Additionally, the invention includes an expression vector comprisingsuch isolated nucleic acids operably linked to the nucleic acid withregulatory sequences effective for expression of the nucleic acid in aselected host cell, for heterologous expression. The host cells can be aeukaryotic cell, a bacterial cell, an insect cell or a yeast cell. Suchcells can be used, for example, in a method of producing a recombinantβ-secretase enzyme, where the method further includes subjecting anextract or cultured medium from said cell to an affinity matrix, such asa matrix formed from a β-secretase inhibitor molecule or antibody, asdetailed herein.

The invention is also directed to a method of screening for compoundsthat inhibit Aβ production, comprising contacting a β-secretasepolypeptide, such as those full-length or truncated forms describedabove, with (i) a test compound and (ii) a β-secretase substrate, andselecting the test compound as capable of inhibiting Aβ production ifthe β-secretase polypeptide exhibits less β-secretase activity in thepresence of than in the absence of the test compound. Such an assay maybe cell-based, with one or both of the enzyme and the substrate producedby the cell, such as the co-expression cell referred to above. Kitsembodying such screening methods also form a part of the invention.

The screening method may further include administering a test compoundto a mammalian subject having Alzheimer's disease or Alzheimer's diseaselike pathology, and selecting the compound as a therapeutic agentcandidate if, following such administration, the subject maintains orimproves cognitive ability or the subject shows reduced plaque burden.Preferably, such a subject is a comprising a transgene for humanβ-amyloid precursor protein (β-APP), such as a mouse bearing a transgenewhich encodes a human β-APP, including a mutant variants thereof, asexemplified in the specification.

In a related embodiment, the invention includes β-secretase inhibitorcompound selected according to the methods described above. Suchcompounds may be is selected, for example, from a phage displayselection system (“library”), such as are known in the art. According toanother aspect, such libraries may be “biased” for the sequence peptideSEQ ID NO: 97 [P10—P4′D→V]. Other inhibitors include, or may be derivedfrom peptide inhibitors herein identified, such as inhibitors SEQ ID NO:78, SEQ ID NO: 72, SEQ ID NO: 78 and SEQ ID NO: 81.

Also forming part of the invention are knock-out mice, characterized byinactivation or deletion of an endogenous β-secretase gene, such asgenes encodes a protein having at least 90% sequence identity to thesequence SEQ ID NO: 65. The deletion or inactivation may be inducible,such as by insertion of a Cre-lox expression system into the mousegenome.

According to a further related aspect, the invention includes a methodof screening for drugs effective in the treatment of Alzheimer's diseaseor other cerebrovascular amyloidosis characterized by Aβ deposition.According to this aspect of the invention, a mammalian subjectcharacterized by overexpression of β-APP and/or deposition of Aβ isgiven a test compound selected for its ability to inhibit β-secretaseactivity a β-secretase protein according to claim 37. The compound isselected as a potential therapeutic drug compound, if it reduces theamount of Aβ deposition in said subject or if it maintains or improvescognitive ability in the subject. According to one preferred embodiment,the mammalian subject is a transgenic mouse bearing a transgene encodinga human β-APP or a mutant thereof.

The invention also includes a method of treating a patient afflictedwith or having a predilection for Alzheimer's disease or othercerebrovascular amyloidosis. According to this aspect, the enzymatichydrolysis of APP to Aβ is blocked by administering to the patient apharmaceutically effective dose of a compound effective to inhibit oneor more of the various forms of the enzyme described herein. Accordingto another feature, the therapeutic compound is derived from a peptideselected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 78, SEQID NO: 81 and SEQ ID NO: 97. Such derivation may be effected by thevarious phage selection systems described herein, in conjunction withthe screening methods of the invention, or other such methods.Alternatively, or in addition, derivation may be achieved via rationalchemistry approaches, including molecular modeling, known in themedicinal chemistry art. Such compounds will preferably be rather potentinhibitors of secretase enzymatic activity, evidenced by a K_(i) of lessthan about 1–50 μM in a MBP-C125sw assay. Such compounds also form thebasis for therapeutic drug compositions in accordance with the presentinvention, which may also include a pharmaceutically effectiveexcipient.

According to yet another related aspect, the invention includes a methodof diagnosing the presence of or a predilection for Alzheimer's diseasein a patient. This method includes detecting the expression level of agene comprising a nucleic acid encoding β-secretase in a cell samplefrom said patient, and diagnosing the patient as having or having apredilection for Alzheimer's disease, if said expression level issignificantly greater than a pre-determined control expression level.Detectable nucleic acids, and primers useful in such detection, aredescribed in detail herein. Such nucleic acids may exclude a nucleicacid encoding the preproenzyme [1–501]. The invention is furtherdirected to method of diagnosing the presence of or a predilection forAlzheimer's disease in a patient, comprising measuring β-secretaseenzymatic activity in a cell sample from said patient, and diagnosingthe patient as having or having a predilection for Alzheimer's disease,if said level enzymatic activity level is significantly greater than apre-determined control activity level. The diagnostic methods may becarried out in a whole cell assay and/or on a nucleic acid derived froma cell sample of said patient.

The invention also includes a method of purifying a β-secretase proteinenzyme molecule. According to this aspect, an impure sample containingβ-secretase enzyme activity with an affinity matrix which includes aβ-secretase inhibitor, such as the various inhibitor molecules describedherein.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the sequence of a polynucleotide (SEQ ID NO: 1) whichencodes human β-secretase translation product shown in FIG. 2A.

FIG. 1B shows the polynucleotide of FIG. 1A, including putative 5′- and3′-untranslated regions (SEQ ID NO: 44).

FIG. 2A shows the amino acid sequence (SEQ ID NO: 2)[1–501] of thepredicted translation product of the open reading frame of thepolynucleotide sequence shown in FIGS. 1A and 1B.

FIG. 2B shows the amino acid sequence of an active fragment of humanβ-secretase (SEQ ID NO: 43)[46–501].

FIG. 3A shows the translation product that encodes an active fragment ofhuman β-secretase, 452stop, (amino acids 1–452 with reference to SEQ IDNO: 2; SEQ ID NO: 59) including a FLAG-epitope tag (underlined; SEQ IDNO: 45) at the C-terminus.

FIG. 3B shows the amino acid sequence of a fragment of human β-secretase(amino acids 46–452 (SEQ ID NO: 58) with reference to SEQ ID NO: 2;including a FLAG-epitope tag (underlined; SEQ ID NO: 45) at theC-terminus.

FIG. 4 shows an elution profile of recombinant β-secretase eluted from agel filtration column.

FIG. 5 shows the full length amino acid sequence of β-secretase 1–501(SEQ ID NO: 2), including the ORF which encodes it (SEQ ID NO: 1), withcertain features indicated, such as “active-D” sites indicating theaspartic acid active catalytic sites, a transmembrane region commencingat position 453, as well as leader (“Signal”) sequence (residues 1–21;SEQ ID NO: 46) and the putative pro region (residues 22–45; SEQ ID NO:47) and where the polynucleotide region corresponding to the activeenzyme portion corresponding to amino acids 46–501 (SEQ ID NO: 43) isshown as SEQ ID NO:42 (nt 135–1503 of SEQ ID NO: 1) and contains aninternal peptide region (SEQ ID NO:56) and a transmembrane region (SEQID NO:62).

FIGS. 6A and 6B show images of silver-stained SDS-PAGE gels on whichpurified β-secretase-containing fractions were run under reducing (6A)and non-reducing (6B) conditions.

FIG. 7 shows a silver-stained SDS-PAGE of β-secretase purified fromheterologous 293T cells expressing the recombinant enzyme.

FIG. 8 shows a silver-stained SDS-PAGE of β-secretase purified fromheterologous Cos A2 cells expressing the recombinant enzyme.

FIG. 9 shows a scheme in which primers derived from the polynucleotide(SEQ ID NO. 76 encoding N-terminus of purified naturally occurringβ-secretase (SEQ ID NO. 77) were used to PCR-clone additional portionsof the molecule, such as fragment SEQ ID NO. 79 encoding by nucleic acidSEQ ID NO. 98, as illustrated.

FIG. 10 shows an alignment of the amino acid sequence of humanβ-secretase (“Human Imapain.seq,” 1–501, SEQ ID NO: 2) compared to(“pBS/mImpain H#3 cons”) consensus mouse sequence: SEQ ID NO: 65.

FIG. 11A shows the nucleotide sequence (SEQ ID NO: 80) of an insert usedin preparing vector pCF.

FIG. 11B shows a linear schematic of pCEK.

FIG. 12 shows a schematic of pCEK.clone 27 used to transfect mammaliancells with β-secretase.

FIG. 13(A–E) shows the nucleotide sequence of pCEK clone 27 (SEQ ID NO:48), with the ORF indicated by the amino acid sequence SEQ ID NO: 2.

FIG. 14A shows a nucleotide sequence inserted into parent vector pcDNA3(SEQ ID NO: 80).

FIG. 14B shows a plot of β-secretase activity in cell lysates from COScells transfected with vectors derived from clones encoding β-secretase.

FIGS. 15A shows an image of an SDS PAGE gel loaded with triplicatesamples of the lysates made from heterologous cells transfected withmutant APP (751 wt) and β-galactosidase as control (lanes d) and fromcells transfected with mutant APP (751 wt) and β-secretase (lanes f)where lanes a, b, and c show lysates from untreated cells, cellstransfected with β-galactosidase alone and cells transfected withβ-secretase alone, respectively, and lane e indicates markers.

FIG. 15B shows an image an image of an SDS PAGE gel loaded withtriplicate samples of the lysates made from heterologous cellstransfected with mutant APP (Swedish mutation) and β-galactosidase ascontrol (lanes c) and from cells transfected with mutant APP (Swedishmutation) and β-secretase (lanes e) where lanes a and b show lysatesfrom cells transfected with β-galactosidase alone and cells transfectedwith β-secretase alone, and lane d indicates markers.

FIGS. 16A and 16B show Western blots of cell supernatants tested forpresence or increase in soluble APP (sAPP).

FIGS. 17A and 17B show Western blots of α-cleaved APP substrate inco-expression cells.

FIG. 18 shows Aβ (x-40) production in 293T cells cotransfected with APPand β-secretase.

FIG. 19A shows a schematic of an APP substrate fragment, and its use inconjunction with antibodies SW192 and 8E-192 in the assay.

FIG. 19B shows the β-secretase cleavage sites in the wild-type APPsequence (SEQ ID NO: 103) and Swedish APP sequence (SEQ ID NO: 104).

FIG. 20 shows a schematic of a second APP substrate fragment derivedfrom APP 638, and it use in conjunction with antibodies SW192 and 8E-192in the assay.

FIG. 21 shows a schematic of pohCK751 vector.

BRIEF DESCRIPTION OF THE SEQUENCES

This section briefly identifies the sequence identification numbersreferred to herein. Number ranges shown in brackets here and throughoutthe specification are referenced to the amino acid sequence SEQ ID NO:2, using conventional N→C-terminus order.

SEQ ID NO: 1 is a nucleic acid sequence that encodes human β-secretase,including an active fragment, as exemplified herein.

SEQ ID NO: 2 is the predicted translation product of SEQ ID NO: 1[1–501].

SEQ ID NOS: 3–21 are degenerate oligonucleotide primers described inExample 1 (Table 4), designed from regions of SEQ ID NO: 2.

SEQ ID NOS: 22–41 are additional oligonucleotide primers used in PCRcloning methods described herein, shown in Table 5.

SEQ ID NO: 42 is a polynucleotide sequence that encodes the activeenzyme β-secretase shown as SEQ ID NO: 43.

SEQ ID NO: 43 is the sequence of an active enzyme portion of humanβ-secretase, the N-terminus of which corresponds to the N-terminus ofthe predominant form of the protein isolated from natural sources[46–501].

SEQ ID NO: 44 is a polynucleotide which encodes SEQ ID NO: 2, including5′ and 3′ untranslated regions.

SEQ ID NO: 45 is the FLAG sequence used in conjunction with certainpolynucleotides.

SEQ ID NO: 46 is the putative leader region of β-secretase [1–21].

SEQ ID NO: 47 is the putative pre-pro region of β-secretase [22–45].

SEQ ID NO: 48 is the sequence of the clone pCEK C1.27 (FIGS. 13A-E).

SEQ ID NO: 49 is a nucleotide sequence of a fragment of the gene whichencodes human β-secretase.

SEQ ID NO: 50 is the predicted translation product of SEQ ID NO: 49.

SEQ ID NO: 51 is a peptide sequence cleavage site of App (swedishmutation).

SEQ ID NOS: 52 and 53 are peptide substrates suitable for use inβ-secretase assays used in the present invention.

SEQ ID NO: 54 is a peptide sequence cleavage site a App (wild type)recognized by Human β-secretase

SEQ ID NO: 55 is amino acids 46–69 of SEQ ID NO: 2.

SEQ ID NO: 56 is an internal peptide just N-terminal to thetransmembrane domain of β-secretase.

SEQ ID NO: 57 is β-secretase [1–419].

SEQ ID NO: 58 is β-secretase [46–452].

SEQ ID NO: 59 is β-secretase [1–452].

SEQ ID NO: 60 is β-secretase [1–420].

SEQ ID NO: 61 is EVM[hydroxyethylene]AEF.

SEQ ID NO: 62 is the amino acid sequence of the transmembrane domain ofβ-secretase shown in (FIG. 5).

SEQ ID NO: 63 is P26-P4′ of APPwt.

SEQ ID NO: 64 is P26-P 1′ of APPwt.

SEQ ID NO: 65 is mouse β-secretase (FIG. 10, lower sequence).

SEQ ID NO: 66 is β-secretase [22–501].

SEQ ID NO: 67 is β-secretase [58–501].

SEQ ID NO: 68 is β-secretase [58–452].

SEQ ID NO: 69 is β-secretase [63–501].

SEQ ID NO: 70 is β-secretase [63–452].

SEQ ID NO: 71 is β-secretase [46–419].

SEQ ID NO: 72 is P10-P4staD→V.

SEQ ID NO: 73 is P4-P4′staD→V.

SEQ ID NO: 74 is β-secretase [22–452].

SEQ ID NO: 75 is β-secretase [63–423].

SEQ ID NO: 76 is nucleic acid encoding the N-terminus of naturallyoccuring β-secretase.

SEQ ID NO: 77 is a peptide fragment at the N-terminus of naturallyoccuring β-secretase.

SEQ ID NO: 78 is a P3-P4′XD→V (VMXVAEF, where X is hydroxyethlene orstatine).

SEQ ID NO: 79 is a peptide fragment of naturally occuring occuringβ-secretase.

SEQ ID NO: 80 is a nucleotide insert in vector pCF used herein.

SEQ ID NO: 81 is P4-P4′XD→V (EVMXVAEF, where X is hydroxyethlene orstatine).

SEQ ID NO: 82 is APP fragment SEVKMDAEF (P5-P4′wt).

SEQ ID NO: 83 is APP fragment SEVNLDAEF (P5-P4′sw).

SEQ ID NO: 84 is APP fragment SEVKLDAEF.

SEQ ID NO: 85 is APP fragment SEVKFDAEF.

SEQ ID NO: 86 is APP fragment SEVNFDAEF.

SEQ ID NO: 87 is APP fragment SEVKMAAEF.

SEQ ID NO: 88 is APP fragment SEVNLAAEF.

SEQ ID NO: 89 is APP fragment SEVKLAAEF.

SEQ ID NO: 90 is APP fragment SEVKLLAEF.

SEQ ID NO: 91 is APP fragment SEVNLLAEF.

SEQ ID NO: 92 is APP fragment SEVKLLAEF.

SEQ ID NO: 93 is APP fragment SEVKFAAEF.

SEQ ID NO: 94 is APP fragment SEVNFLAAEF.

SEQ ID NO: 95 is APP fragment SEVKFLAEF.

SEQ ID NO: 96 is APP fragment SEVNFLAEF.

SEQ ID NO: 97 is APP-derived fragment P10-P4′(D→V): KTEEISEVNLVAEF

SEQ ID NO: 98 is a nucleic acid fragment (FIG. 9).

SEQ ID NO: 99 is the N terminal peptide sequence of β-secretase isolatedfrom human brain, recombinant 293T cells and recombinant Cos A2 cells(Table 3).

SEQ ID NO: 100 is the N terminal peptide sequence of a form ofβ-secretase isolated from recombinant 293T cells.

SEQ ID NO: 101 is the N terminal peptide sequence of a form ofβ-secretase isolated from recombinant 293T cells.

SEQ ID NO: 102 is the N terminal peptide sequence of a form ofβ-secretase isolated from recombinant CosA2 cells.

SEQ ID NO: 103 is the β-secretase cleavage sites in the wild-type APPsequence.

SEQ ID NO: 104 is the β-secretase cleavage sites in the Swedish APP

Detailed Description of the Invention

I. Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art of the present invention.Practitioners are particularly directed to Sambrook, et al. (1989)Molecular Cloning: A Laboratory Manual (Second Edition), Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M., et al. (1998) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., fordefinitions, terms of art and standard methods known in the art ofmolecular biology, particularly as it relates to the cloning protocolsdescribed herein. It is understood that this invention is not limited tothe particular methodology, protocols, and reagents described, as thesemay be varied to produce the same result.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to a polymeric molecule having a backbone that supportsbases capable of hydrogen bonding to typical polynucleotides, where thepolymer backbone presents the bases in a manner to permit such hydrogenbonding in a sequence specific fashion between the polymeric moleculeand a typical polynucleotide (e.g., single-stranded DNA). Such bases aretypically inosine, adenosine, guanosine, cytosine, uracil and thymidine.Polymeric molecules include double and single stranded RNA and DNA, andbackbone modifications thereof, for example, methylphosphonate linkages.

The term “vector” refers to a polynucleotide having a nucleotidesequence that can assimilate new nucleic acids, and propagate those newsequences in an appropriate host. Vectors include, but are not limitedto recombinant plasmids and viruses. The vector (e.g., plasmid orrecombinant virus) comprising the nucleic acid of the invention can bein a carrier, for example, a plasmid complexed to protein, a plasmidcomplexed with lipid-based nucleic acid transduction systems, or othernon-viral carrier systems.

The term “polypeptide” as used herein refers to a compound made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” may be synonymous with the term “polypeptide” or may refer toa complex of two or more polypeptides.

The term “modified”, when referring to a polypeptide of the invention,means a polypeptide which is modified either by natural processes, suchas processing or other post-translational modifications, or by chemicalmodification techniques which are well known in the art. Among thenumerous known modifications which may be present include, but are notlimited to, acetylation, acylation, amidation, ADP-ribosylation,glycosylation, GPI anchor formation, covalent attachment of a lipid orlipid derivative, methylation, myristlyation, pegylation, prenylation,phosphorylation, ubiqutination, or any similar process.

The term “β-secretase” is defined in Section III, herein.

The term “biologically active” used in conjunction with the termβ-secretase refers to possession of a β-secretase enzyme activity, suchas the ability to cleave β-amyloid precursor protein (APP) to produceβ-amyloid peptide (Aβ).

The term “fragment,” when referring to β-secretase of the invention,means a polypeptide which has an amino acid sequence which is the sameas part of but not all of the amino acid sequence of full-lengthβ-secretase polypeptide. In the context of the present invention, thefull length β-secretase is generally identified as SEQ ID NO: 2, the ORFof the full-length nucleotide; however, according to a discovery of theinvention, the naturally occurring active form is probably one or moreN-terminal truncated versions, such as amino acids 46–501 (SEQ IDNO:43), 22–501 (SEQ ID NO:66), 58–501 (SEQ ID NO:67) or 63–501 (SEQ IDNO:69); other active forms are C-terminal truncated forms ending betweenabout amino acids 450 and 452. The numbering system used throughout isbased on the numbering of the sequence SEQ ID NO: 2.

An “active fragment” is a β-secretase fragment that retains at least oneof the functions or activities of β-secretase, including but not limitedto the β-secretase enzyme activity discussed above and/or ability tobind to the inhibitor substrate described herein as P10—P4′staD->V (SEQID NO:72). Fragments contemplated include, but are not limited to, aβ-secretase fragment which retains the ability to cleave β-amyloidprecursor protein to produce β-amyloid peptide. Such a fragmentpreferably includes at least 350, and more preferably at least 400,contiguous amino acids or conservative substitutions thereof ofβ-secretase, as described herein. More preferably, the fragment includesactive aspartyl acid residues in the structural proximities identifiedand defined by the primary polypeptide structure shown as SEQ ID NO: 2and also denoted as “Active-D” sites herein.

A “conservative substitution” refers to the substitution of an aminoacid in one class by an amino acid in the same class, where a class isdefined by common physicochemical amino acid sidechain properties andhigh substitution frequencies in homologous proteins found in nature (asdetermined, e.g., by a standard Dayhoff frequency exchange matrix orBLOSUM matrix). Six general classes of amino acid sidechains,categorized as described above, include: Class I (Cys); Class II (Ser,Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg,Lys); Class V (lIe, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). Forexample, substitution of an Asp for another class III residue such asAsn, Gln, or Glu, is considered to be a conservative substitution.

“Optimal alignment” is defined as an alignment giving the highestpercent identity score. Such alignment can be performed using a varietyof commercially available sequence analysis programs, such as the localalignment program LALIGN using a ktup of 1, default parameters and thedefault PAM. A preferred alignment is the pairwise alignment using theCLUSTAL-W program in MacVector, operated with default parameters,including an open gap penalty of 10.0, an extended gap penalty of 0.1,and a BLOSUM30 similarity matrix.

“Percent sequence identity,” with respect to two amino acid orpolynucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 80% amino acid sequence identity means that 80% of the amino acidsin two or more optimally aligned polypeptide sequences are identical. Ifa gap needs to be inserted into a first sequence to optimally align itwith a second sequence, the percent identity is calculated using onlythe residues that are paired with a corresponding amino acid residue(i.e., the calculation does not consider residues in the secondsequences that are in the “gap” of the first sequence.

A first polypeptide region is said to “correspond” to a secondpolypeptide region when the regions are essentially co-extensive whenthe sequences containing the regions are aligned using a sequencealignment program, as above. Corresponding polypeptide regions typicallycontain a similar, if not identical, number of residues. It will beunderstood, however, that corresponding regions may contain insertionsor deletions of residues with respect to one another, as well as somedifferences in their sequences.

A first polynucleotide region is said to “correspond” to a secondpolynucleotide region when the regions are essentially co-extensive whenthe sequences containing the regions are aligned using a sequencealignment program, as above. Corresponding polynucleotide regionstypically contain a similar, if not identical, number of residues. Itwill be understood, however, that corresponding regions may containinsertions or deletions of bases with respect to one another, as well assome differences in their sequences.

The term “sequence identity” means nucleic acid or amino acid sequenceidentity in two or more aligned sequences, aligned as defined above.

“Sequence similarity” between two polypeptides is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.Thus, 80% protein sequence similarity means that 80% of the amino acidresidues in two or more aligned protein sequences are conserved aminoacid residues, i.e. are conservative substitutions.

“Hybridization” includes any process by which a strand of a nucleic acidjoins with a complementary nucleic acid strand through base pairing.Thus, strictly speaking, the term refers to the ability of thecomplement of the target sequence to bind to the test sequence, orvice-versa.

“Hybridization conditions” are based in part on the melting temperature(Tm) of the nucleic acid binding complex or probe and are typicallyclassified by degree of “stringency” of the conditions under whichhybridization is measured. The specific conditions that define variousdegrees of stringency (i.e., high, medium, low) depend on the nature ofthe polynucleotide to which hybridization is desired, particularly itspercent GC content, and can be determined empirically according tomethods known in the art. Functionally, maximum stringency conditionsmay be used to identify nucleic acid sequences having strict identity ornear-strict identity with the hybridization probe; while high stringencyconditions are used to identify nucleic acid sequences having about 80%or more sequence identity with the probe.

The term “gene” as used herein means the segment of DNA involved inproducing a polypeptide chain; it may include regions preceding andfollowing the coding region, e.g. 5′ untranslated (5′ UTR) or “leader”sequences and 3′ UTR or “trailer” sequences, as well as interveningsequences (introns) between individual coding segments (exons).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Such isolatedpolynucleotides may be part of a vector and/or such polynucleotides orpolypeptides may be part of a composition, such as a recombinantlyproduced cell (heterologous cell) expressing the polypeptide, and stillbe isolated in that such vector or composition is not part of itsnatural environment.

An “isolated polynucleotide having a sequence which encodes β-secretase”is a polynucleotide that contains the coding sequence of β-secretase, oran active fragment thereof, (i) alone, (ii) in combination withadditional coding sequences, such as fusion protein or signal peptide,in which the β-secretase coding sequence is the dominant codingsequence, (iii) in combination with non-coding sequences, such asintrons and control elements, such as promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in a suitable host, and/or (iv) in a vector or hostenvironment in which the β-secretase coding sequence is a heterologousgene.

The terms “heterologous DNA,” “heterologous RNA,” “heterologous nucleicacid,” “heterologous gene,” and “heterologous polynucleotide” refer tonucleotides that are not endogenous to the cell or part of the genome inwhich they are present; generally such nucleotides have been added tothe cell, by transfection, microinjection, electroporation, or the like.Such nucleotides generally include at least one coding sequence, butthis coding sequence need not be expressed.

The term “heterologous cell” refers to a recombinantly produced cellthat contains at least one heterologous DNA molecule.

A “recombinant protein” is a protein isolated, purified, or identifiedby virtue of expression in a heterologous cell, said cell having beentransduced or transfected, either transiently or stably, with arecombinant expression vector engineered to drive expression of theprotein in the host cell.

The term “expression” means that a protein is produced by a cell,usually as a result of transfection of the cell with a heterologousnucleic acid.

“Co-expression” is a process by which two or more proteins or RNAspecies of interest are expressed in a single cell. Co-expression of thetwo or more proteins is typically achieved by transfection of the cellwith one or more recombinant expression vectors(s) that carry codingsequences for the proteins. In the context of the present invention, forexample, a cell can be said to “co-express” two proteins, if one or bothof the proteins is heterologous to the cell.

The term “expression vector” refers to vectors that have the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are commerciallyavailable. Selection of appropriate expression vectors is within theknowledge of those having skill in the art.

The terms “purified” or “substantially purified” refer to molecules,either polynucleotides or polypeptides, that are removed from theirnatural environment, isolated or separated, and are at least 90% andmore preferably at least 95–99% free from other components with whichthey are naturally associated. The foregoing notwithstanding, such adescriptor does not preclude the presence in the same sample of splice-or other protein variants (glycosylation variants) in the same,otherwise homogeneous, sample.

A protein or polypeptide is generally considered to be “purified toapparent homogeneity” if a sample containing it shows a single proteinband on a silver-stained polyacrylamide electrophoretic gel.

The term “crystallized protein” means a protein that has co-precipitatedout of solution in pure crystals consisting only of the crystal, butpossibly including other components that are tightly bound to theprotein.

A “variant” polynucleotide sequence may encode a “variant” amino acidsequence that is altered by one or more amino acids from the referencepolypeptide sequence. The variant polynucleotide sequence may encode avariant amino acid sequence, which contains “conservative”substitutions, wherein the substituted amino acid has structural orchemical properties similar to the amino acid which it replaces. Inaddition, or alternatively, the variant polynucleotide sequence mayencode a variant amino acid sequence, which contains “non-conservative”substitutions, wherein the substituted amino acid has dissimilarstructural or chemical properties to the amino acid which it replaces.Variant polynucleotides may also encode variant amino acid sequences,which contain amino acid insertions or deletions, or both. Furthermore,a variant polynucleotide may encode the same polypeptide as thereference polynucleotide sequence but, due to the degeneracy of thegenetic code, has a polynucleotide sequence that is altered by one ormore bases from the reference polynucleotide sequence.

An “allelic variant” is an alternate form of a polynucleotide sequence,which may have a substitution, deletion or addition of one or morenucleotides that does not substantially alter the function of theencoded polypeptide.

“Alternative splicing” is a process whereby multiple polypeptideisoforms are generated from a single gene, and involves the splicingtogether of nonconsecutive exons during the processing of some, but notall, transcripts of the gene. Thus, a particular exon may be connectedto any one of several alternative exons to form messenger RNAs. Thealternatively-spliced mRNAs produce polypeptides (“splice variants”) inwhich some parts are common while other parts are different.

“Splice variants” of β-secretase, when referred to in the context of anmRNA transcript, are mRNAs produced by alternative splicing of codingregions, i.e., exons, from the β-secretase gene.

“Splice variants” of β-secretase, when referred to in the context of theprotein itself, are β-secretase translation products that are encoded byalternatively-spliced β-secretase mRNA transcripts.

A “mutant” amino acid or polynucleotide sequence is a variant amino acidsequence, or a variant polynucleotide sequence, which encodes a variantamino acid sequence that has significantly altered biological activityor function from that of the naturally occurring protein.

A “substitution” results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

The term “modulate” as used herein refers to the change in activity ofthe polypeptide of the invention. Modulation may relate to an increaseor a decrease in biological activity, binding characteristics, or anyother biological, functional, or immunological property of the molecule.

The terms “antagonist” and “inhibitor” are used interchangeably hereinand refer to a molecule which, when bound to the polypeptide of thepresent invention, modulates the activity of enzyme by blocking,decreasing, or shortening the duration of the biological activity. Anantagonist as used herein may also be referred to as a “β-secretaseinhibitor” or “β-secretase blocker.” Antagonists may themselves bepolypeptides, nucleic acids, carbohydrates, lipids, small molecules(usually less than 1000 kD), or derivatives thereof, or any other ligandwhich binds to and modulates the activity of the enzyme.

β-Secretase Compositions

The present invention provides an isolated, active human β-secretaseenzyme, which is further characterized as an aspartyl (aspartic)protease or proteinase, optionally, in purified form. As defined morefully in the sections that follow, β-secretase exhibits a proteolyticactivity that is involved in the generation of β-amyloid peptide fromβ-amyloid precursor protein (APP), such as is described in U.S. Pat. No.5,744,346, incorporated herein by reference. Alternatevely, or inaddition, the β-secretase is characterized by its ability to bind, withmoderately high affinity, to an inhibitor substrate described herein asP10—P4′ staD→V (SEQ ID NO.: 72). According to an important feature ofthe present invention, a human form of β-secretase has been isolated,and its naturally occurring form has been characterized, purified andsequenced.

According to another aspect of the invention, nucleotide sequencesencoding the enzyme have been identified. In addition, the enzyme hasbeen further modified for expression in altered forms, such as truncatedforms, which have similar protease activity to the naturally occurringor full length recombinant enzyme. Using the information providedherein, practitioners can isolate DNA encoding various active forms ofthe protein from available sources and can express the proteinrecombinantly in a convenient expression system. Alternatively and inaddition, practitioners can purify the enzyme from natural orrecombinant sources and use it in purified form to further characterizeits structure and function. According to a further feature of theinvention, polynucleotides and proteins of the invention areparticularly useful in a variety of screening assay formats, includingcell-based screening for drugs that inhibit the enzyme. Examples of usesof such assays, as well as additional utilities for the compositions areprovided in Section IV, below.

β-secretase is of particular interest due to its activity andinvolvement in generating fibril peptide components that are the majorcomponents of amyloid plaques in the central nervous system (CNS), suchas are seen in Alzheimer's disease, Down's syndrome and other CNSdisorders. Accordingly, a useful feature of the present inventionincludes an isolated form of the enzyme that can be used, for example,to screen for inhibitory substances which are candidates fortherapeutics for such disorders.

A. Isolation of Polynucleotides encoding Human β-secretasePolynucleotides encoding human β-secretase were obtained by PCR cloningand hybridization techniques as detailed in Examples 1–3 and describedbelow. FIG. 1A shows the sequence of a polynucleotide (SEQ ID NO: 1)which encodes a form of human β-secretase (SEQ ID NO.: 2 [1–501].Polynucleotides encoding human β-secretase are conveniently isolatedfrom any of a number of human tissues, preferably tissues of neuronalorigin, including but not limited to neuronal cell lines such as thecommercially available human neuroblastoma cell line IMR-32 availablefrom the American Type Culture Collection (Manassas, Va.; ATTC CCL 127)and human fetal brain, such as a human fetal brain cDNA libraryavailable from OriGene Technologies, Inc. (Rockville, Md.).

Briefly, human β-secretase coding regions were isolated by methods wellknown in the art, using hybridization probes derived from the codingsequence provided as SEQ ID NO: 1. Such probes can be designed and madeby methods well known in the art. Exemplary probes, including degenerateprobes, are described in Example 1. Alternatively, a cDNA library isscreened by PCR, using, for example, the primers and conditionsdescribed in Example 2 herein. Such methods are discussed in more detailin Part B, below.

cDNA libraries were also screened using a 3′-RACE (Rapid Amplificationof cDNA Ends) protocol according to methods well known in the art(White, B. A., ed., PCR Cloning Protocols; Humana Press, Totowa, N.J.,1997; shown schematically in FIG. 9). Here primers derived from the 5′portion of SEQ ID NO: 1 are added to partial cDNA substrate clone foundby screening a fetal brain cDNA library as described above. Arepresentative 3′RACE reaction used in determining the longer sequenceis detailed in Example 3 and is described in more detail in Part B,below.

Human β-secretase, as well as additional members of the neuronalaspartyl protease family described herein may be identified by the useof random degenerate primers designed in accordance with any portion ofthe polypeptide sequence shown as SEQ ID NO: 2. For example, inexperiments carried out in support of the present invention, anddetailed in Example 1 herein, eight degenerate primer pools, each 8-folddegenerate, were designed based on a unique 22 amino acid peptide regionselected from SEQ ID: 2. Such techniques can be used to identify furthersimilar sequences from other species and/or representing other membersof this protease family.

Preparation of Polynucleotides

The polynucleotides described herein may be obtained by screening cDNAlibraries using oligonucleotide probes, which can hybridize to and/orPCR-amplify polynucleotides that encode human β-secretase, as disclosedabove. cDNA libraries prepared from a variety of tissues arecommercially available, and procedures for screening and isolating cDNAclones are well known to those of skill in the art. Genomic librariescan likewise be screened to obtain genomic sequences includingregulatory regions and introns. Such techniques are described in, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, FM et al. (1998) Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y.

The polynucleotides may be extended to obtain upstream and downstreamsequences such as promoters, regulatory elements, and 5′ and 3′untranslated regions (UTRs). Extension of the available transcriptsequence may be performed by numerous methods known to those of skill inthe art, such as PCR or primer extension (Sambrook et al. supra), or bythe RACE method using, for example, the MARATHON RACE kit (Cat. #K1802-1; Clontech, Palo Alto, Calif.).

Alternatively, the technique of “restriction-site” PCR (Gobinda et al.(1993) PCR Methods Applic. 2:318–22), which uses universal primers toretrieve flanking sequence adjacent a known locus, may be employed togenerate additional coding regions. First, genomic DNA is amplified inthe presence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia T et al. (1988) Nucleic AcidsRes 16:8186). The primers may be designed using OLIGO(R) 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth, Minn.), oranother appropriate program, to be 22–30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68–72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template.

Capture PCR (Lagerstrom M et a. (1991) PCR Methods Applic 1:111–19) is amethod for PCR amplification of DNA fragments adjacent to a knownsequence in human and yeast artificial chromosome DNA. Capture PCR alsorequires multiple restriction enzyme digestions and ligations to placean engineered double-stranded sequence into a flanking part of the DNAmolecule before PCR.

Another method which may be used to retrieve flanking sequences is thatof Parker, J D et al. (1991; Nucleic Acids Res 19:3055–60).Additionally, one can use PCR, nested primers and PromoterFinder(™)libraries to “walk in” genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. Preferred libraries for screening for full lengthcDNAs are ones that have been size-selected to include larger cDNAs.Also, random primed libraries are preferred in that they will containmore sequences which contain the 5′ and upstream regions of genes. Arandomly primed library may be particularly useful if an oligo d(T)library does not yield a full-length cDNA. Genomic libraries are usefulfor extension into the 5′ nontranslated regulatory region.

The polynucleotides and oligonucleotides of the invention can also beprepared by solid-phase methods, according to known synthetic methods.Typically, fragments of up to about 100 bases are individuallysynthesized, then joined to form continuous sequences up to severalhundred bases.

B. Isolation of β-Secretase

The amino acid sequence for a full-length human β-secretase translationproduct is shown as SEQ ID NO: 2 in FIG. 2A. According to the discoveryof the present invention, this sequence represents a “pre pro” form ofthe enzyme that was deduced from the nucleotide sequence informationdescribed in the previous section in conjunction with the methodsdescribed below. Comparison of this sequence with sequences determinedfrom the biologically active form of the enzyme purified from naturalsources, as described in Part 4, below, indicate that it is likely thatan active and predominant form of the enzyme is represented by sequenceshown in FIG. 2B (SEQ ID NO: 43), in which the first 45 amino acids ofthe open-reading frame deduced sequence have been removed. This suggeststhat the enzyme may be post-translationally modified by proteolyticactivity, which may be autocatalytic in nature. Further analysis,illustrated by the schematics shown in FIG. 5 herein, indicates that theenzyme contains a hydrophobic, putative transmembrane region near itsC-terminus. As described below, a further discovery of the presentinvention is that the enzyme can be truncated prior to thistransmembrane region and still retain β-secretase activity.

1. Purification of β-Secretase from Natural and Recombinant Sources

According to an important feature of the present invention, β-secretasehas now been purified from natural and recombinant sources. U.S. Pat.No. 5,744,346, incorporated herein by reference, describes isolation ofβ-secretase in a single peak having an apparent molecular weight of260–300,000 (Daltons) by gel exclusion chromatography. It is a discoveryof the present invention that the native enzyme can be purified toapparent homogeneity by affinity column chromatography. The methodsrevealed herein have been used on preparations from brain tissue as wellas on preparations from 293T and recombinant cells; accordingly, thesemethods are believed to be generally applicable over a variety of tissuesources. The practitioner will realize that certain of the preparationsteps, particularly the initial steps, may require modification toaccommodate a particular tissue source and will adapt such proceduresaccording to methods known in the art. Methods for purifying β-secretasefrom human brain as well as from cells are detailed in Example 5.Briefly, cell membranes or brain tissue are homogenized, fractionated,and subjected to various types of column chromatographic matrices,including wheat germ agglutinin-agarose (WGA), anion exchangechromatography and size exclusion. Activity of fractions can be measuredusing any appropriate assay for β-secretase activity, such as theMBP-C125 cleavage assay detailed in Example 4. Fractions containingβ-secretase activity elute from this column in a peak elution volumecorresponding to a size of about 260–300 kilodaltons.

The foregoing purification scheme, which yields approximately 1,500-foldpurification, is similar to that described in detail in U.S. Pat. No.5,744,346, incorporated herein by reference. In accordance with thepresent invention, further purification can be achieved by applying thecation exchange flow-through material to an affinity column that employsas its affinity matrix a specific inhibitor of β-secretase, termed“P10-P4′staD→V” (NH₂-KTEEISEVN[sta]VAEF-CO₂H; SEQ ID NO.: 72). Thisinhibitor, and methods for making a Sepharose affinity column whichincorporates it, are described in Example 7. After washing the column,β-secretase and a limited number of contaminating proteins were elutedwith pH 9.5 borate buffer. The eluate was then fractionated by anionexchange HPLC, using a Mini-Q column. Fractions containing the activitypeak were pooled to give the final β-secretase preparation. Results ofan exemplary run using this purification scheme are summarized inTable 1. FIG. 6A shows a picture of a silver-stained SDS PAGE gel rununder reducing conditions, in which β-secretase runs as a 70 kilodaltonband. The same fractions run under non-reducing conditions (FIG. 6B)provide evidence for disulfide cross-linked oligomers. When the anionexchange pool fractions 18–21 (see FIG. 6B) were treated withdithiothreitol (DTT) and re-chromatographed on a Mini Q column, thensubjected to SDS-PAGE under non-reducing conditions, a single bandrunning at about 70 kilodaltons was observed. Surprisingly, the purityof this preparation is at least about 200 fold higher than thepreviously purified material, described in U.S. Pat. No. 5,744,346. Byway of comparison, the most pure fraction described therein exhibited aspecific activity of about 253 nM/μg protein, taking into considerationthe MW of substrate MBP-C26sw (45 kilodaltons). The present methodtherefore provides a preparation that is at least about 1000-fold higherpurity (affinity eluate) and as high as about 6000-fold higher puritythan that preparation, which represented at least 5 to 1000-fold higherpurity than the enzyme present in a solubilized but unenriched membranefraction from human 293 cells.

TABLE 1 Preparation of β-secretase from Human Brain Total SpecificActivity^(a) Activity^(b) Purification nM/h nM/h/μg prot. % Yield (fold)Brain Extract 19,311,150 4.7 100 1 WGA Eluate 21,189,600 81.4 110 17Affinity Eluate 11,175,000 257,500 53 54,837 Anion Exchange 3,267,6851,485,312 17 316,309 Pool ^(a)Activity in MBP-C125sw assay${{\,^{b}{Specific}}\mspace{14mu}{Activity}} = \frac{\left( {{Product}\mspace{14mu}{{conc}.\mspace{14mu}{nM}}} \right)\left( {{Dilution}\mspace{14mu}{factor}} \right)}{\begin{matrix}{\left( {{Enzyme}\mspace{14mu}{{sol}.\mspace{14mu}{vol}}} \right)\left( {{{Incub}.\mspace{14mu}{time}}\mspace{14mu} h} \right)} \\\left( {{Enzyme}\mspace{14mu}{{conc}.\mspace{14mu}\mu}\; g\text{/}{vol}} \right)\end{matrix}}$

Example 5 also describes purification schemes used for purifyingrecombinant materials from heterologous cells transfected with theβ-secretase coding sequence. Results from these purifications areillustrated in FIGS. 7 and 8. Further experiments carried out in supportof the present invention, showed that the recombinant material has anapparent molecular weight in the range from 260,000 to 300,000 Daltonswhen measured by gel exclusion chromatography. FIG. 4 shows an activityprofile of this preparation run on a gel exclusion chromatographycolumn, such as a Superdex 200 (26/60) column, according to the methodsdescribed in U.S. Pat. No. 5,744,346, incorporated herein by reference.

1. Sequencing of β-secretase Protein

A schematic overview summarizing methods and results for determining thecDNA sequence encoding the N-terminal peptide sequence determined frompurified β-secretase is shown in FIG. 9. N-terminal sequencing ofpurified β-secretase protein isolated from natural sources yielded a21-residue peptide sequence, SEQ ID NO. 77, as described above. Thispeptide sequence, and its reverse translated fully degenerate nucleotidesequence, SEQ ID NO. 76, is shown in the top portion of FIG. 4. Twopartially degenerate primer sets used for RT-PCR amplification of a cDNAfragment encoding this peptide are also summarized in FIG. 4. Primer set1 consisted of DNA nucleotide primers #3427–3434, shown in Table 3(Example 3). Matrix RT-PCR using combinations of primers from this setwith cDNA reverse transcribed from primary human neuronal cultures astemplate yielded the predicted 54 bp cDNA product with primers#3428–3433, also described in Table 3.

In further experiments carried out in support of the present invention,it was found that oligonucleotides from primer sets 1 and 2 could alsobe used to amplify cDNA fragments of the predicted size from mouse brainmRNA. DNA sequence demonstrated that such primers could also be used toclone the murine homolog(s) and other species homologs of humanβ-secretase and/or additional members of the aspartyl protease familydescribed herein by standard RACE-PCR technology. The sequence of amurine homolog is presented in FIG. 10 (lower sequence; “pBS/MuImPainH#3 cons”); SEQ ID NO. 65. The murine polypeptide sequence is about 95%identical to the human polypeptide sequence.

2. 5′ and 3′ RACE-PCR for Additional Sequence, Cloning, and mRNAAnalysis

The unambiguous internal nucleotide sequence from the amplified fragmentprovided information which facilitated the design of internal primersmatching the upper (coding) strand for 3′ RACE, and lower (non-coding)strand for 5′ RACE (Frohman, M. A., M. K. Dush and G. R. Martin (1988).“Rapid production of full-length cDNAs from rare transcripts:amplification using a single gene specific oligo-nucleotide primer.”Proc. Natl. Acad. Sci. U.S.A. 85(23): 8998–9002.) The DNA primers usedfor this experiment (#3459 & #3460) are illustrated schematically inFIG. 9, and the exact sequence of these primers is presented in Table 4of Example 3.

Primers #3459 and #3476 (Table 5) were used for initial 3′ RACEamplification of downstream sequences from the IMR-32 cDNA library inthe vector pLPCXlox. The library had previously been sub-divided into100 pools of 5,000 clones per pool, and plasmid DNA was isolated fromeach pool. A survey of the 100 pools with the primers described in Part2, above, identified individual pools containing β-secretase clones fromthe library. Such clones can be used for RACE-PCR analysis.

An approximately 1.8 Kb PCR fragment was observed by agarose gelfractionation of the reaction products. The PCR product was purifiedfrom the gel and subjected to DNA sequence analysis using primer #3459(Table 5). The resulting clone sequence, designated 23A, was determined.Six of the first seven deduced amino-acids from one of the readingframes of 23A were an exact match with the last 7 amino-acids of theN-terminal sequence (SEQ ID NO. 77) determined from the purified proteinisolated from natural sources in other experiments carried out insupport of this invention. This observation provided internal validationof the sequences, and defined the proper reading frame downstream.Furthermore, this DNA sequence facilitated design of additional primersfor extending the sequence further downstream, verifying the sequence bysequencing the opposite strand in the upstream direction, and furtherfacilitated isolating the cDNA clone.

A DNA sequence of human β-secretase is illustrated as SEQ ID NO: 42corresponding to SEQ ID NO: 1 including 5′- and 3′-untranslated regions.This sequence was determined from a partial cDNA clone (9C7e.35)isolated from a commercially available human fetal brain cDNA librarypurchased from OriGene™, the 3′ RACE product 23A, and additionalclones—a total of 12 independent cDNA clones were used to determine thecomposite sequence. The composite sequence was assembled by sequencingoverlapping stretches of DNA from both strands of the clone or PCRfragment. The predicted full length translation product is shown as SEQID NO: 2 in FIG. 1B.

4. Tissue Distribution of β-secretase and Related Transcripts

Oligonucleotide primer #3460 (SEQ ID NO. 39, Table 5) was employed as anend-labeled probe on Northern blots to determine the size of thetranscript encoding β-secretase and to examine its expression in IMR-32cells. Additional primers were used to isolate the mouse cDNA and tocharacterize mouse tissues, using Marathon RACE ready cDNA preparations(Clontech, Palo Alto, Calif.). TABLE 2 summarizes the results ofexperiments in which various human and murine tissues were tested forthe presence of β-secretase-encoding transcripts by PCR or Northernblotting.

For example, the oligo-nucleotide probe 3460 (SEQ ID NO: 39) hybridizedto a 2 Kb transcript in IMR-32 cells, indicating that the mRNA encodingthe β-secretase enzyme is 2 Kb in size in this tissue. Northern blotanalysis of total RNA isolated from the human T-cell line Jurkat, andhuman myclomonocyte line Thp1 with the 3460 oligo-nucleotide probe 3460also revealed the presence of a 2 kb transcript in these cells.

The oligonucleotide probe #3460 also hybridizes to a ˜2 kb transcript inNorthern blots containing RNA from all human organs examined to date,from both adult and fetal tissue. The organs surveyed include heart,brain, liver, pancreas, placenta, lung, muscle, uterus, bladder, kidney,spleen, skin, and small intestine. In addition, certain tissues, e.g.pancreas, liver, brain, muscle, uterus, bladder, kidney, spleen andlung, show expression of larger transcripts of ˜4.5 kb, 5 kb, and 6.5 kbwhich hybridize with oligonucleotide probe #3460.

In further experiments carried out in support of the present invention,Northern blot results were obtained with oligonucleotide probe #3460 byemploying a riboprobe derived from SEQ ID NO: 1, encompassingnucleotides #155–1014. This clone provides an 860 bp riboprobe,encompassing the catalytic domain-encoding portion of β-secretase, forhigh stringency hybridization. This probe hybridized with highspecificity to the exact match mRNA expressed in the samples beingexamined. Northern blots of mRNA isolated from IMR-32 and 1°HNC probedwith this riboprobe revealed the presence of the 2 kb transcriptpreviously detected with oligonucleotide #3460, as well as a novel,higher MW transcript of ˜5 kb. Hybridization of RNA from adult and fetalhuman tissues with this 860 nt riboprobe also confirmed the resultobtained with the oligonucleotide probe #3460. The mRNA encodingβ-secretase is expressed in all tissues examined, predominantly as an ˜5kb transcript. In adult, its expression appeared lowest in brain,placenta, and lung, intermediate in uterus, and bladder, and highest inheart, liver, pancreas, muscle, kidney, spleen, and lung. In fetaltissue, the message is expressed uniformly in all tissues examined.

TABLE 2 Tissue distribution of human and murine β-secretase transcriptsSize Messages Found (Kb): Human Mouse Tissue/Organ Heart 2^(a) 3.5, 3.8,5 & 7 Brain 2, 3, 4, and 7 3.5, 3.8, 5 & 7 Liver 2, 3, 4, and 7 3.5,3.8, 5 & 7 Pancreas 2, 3, 4, and 7 nd^(d) Placenta 2^(a), 4 and 7^(b) ndLung 2^(a), 4 and 7^(b) 3.5, 3.8, 5 & 7 Muscle 2^(a) and 7^(b) 3.5, 3.8,5 & 7 Uterus 2^(a), 4, and 7 nd Bladder 2^(a), 3, 4, and 7 nd Kidney2^(a), 3, 4, and 7 3.5, 3.8, 5 & 7 Spleen 2^(a), 3, 4, and 7 nd Testisnd 4.5 Kb, 2 Kb Stomach nd 5^(a) Sm. Intestine nd 3.5, 3.8, 5 & 7 fBrain 2^(a), 3, 4, and 7 nd f Liver 2^(a), 3, 4, and 7 nd f Lung 2^(a),3, 4, and 7 nd f Muscle 2^(a), 3, 4, and 7 nd f Heart 2^(a), 3, 4, and 7nd f Kidney 2^(a), 3, 4, and 7 nd f Skin 2^(a), 3, 4, and 7 nd f Sm.Intestine 2^(a), 3, 4, and 7 nd Cell Line IMR32 2^(a), 5 & 7 U937 2^(a)THP1 2^(a) Jurkat 2^(a) HL60 none A293 5 & 7 NALM6 5 & 7 A549 5 & 7 Hela2, 4, 5, & 7 PC12 2 & 5 J774 5 Kb, 2 Kb P388D1 ccl46 5 Kb (very little),2 Kb P19 5 Kb, 2 Kb RBL 5 Kb, 2 Kb EL4 5 Kb, 2 Kb Clontech Human Brainregion Tissue/Organ Cerebellum 2 Kb, 4 Kb, 6 Kb Cerebral Cx 2 Kb, 4 Kb,6 Kb Medulla 2 Kb, 4 Kb, 6 Kb Spinal Cord 2 Kb, 4 Kb, 6 Kb OccipitalPole 2 Kb, 4 Kb, 6 Kb Frontal Lobe 2 Kb, 4 Kb, 6 Kb Amygdala 2 Kb, 4 Kb,6 Kb Caudate N. 2 Kb, 4 Kb, 6 Kb Corpus Callosum 2 Kb, 4 Kb, 6 KbHippocampus 2 Kb, 4 Kb, 6 Kb Substantia Nigra 2 Kb, 4 Kb, 6 Kb Thalamus2 Kb, 4 Kb, 6 Kb ^(a)by oligo 3460 probe only ^(b)faint ^(c)f = fetal^(d)nd = not determined

5. Active Forms of β-secretase

a. N-terminus

The full-length open reading frame (ORF) of human β-secretase isdescribed above, and its sequence is shown in FIG. 2A as SEQ ID NO: 2.However, as mentioned above, a further discovery of the presentinvention indicates that the predominant form of the active, naturallyoccurring molecule is truncated at the N-terminus by about 45 aminoacids. That is, the protein purified from natural sources was N-terminalsequenced according to methods known in the art (Argo Bioanalytica,Morris Plains, N.J.). The N-terminus yielded the following sequence:EGDEEPEEPGRRGSFVEMVDNLRG . . . (SEQ ID NO: 55). This corresponds toamino acids 46–69 of the ORF-derived putative sequence. Based on thisobservation and others described below, the N-terminus of an active,naturally occurring, predominant human brain form of the enzyme is aminoacid 46, with respect to SEQ ID NO: 2. Further processing of thepurified protein provided the sequence of an internal peptide:IGFAVSACHVHDEFR (SEQ ID NO: 56), which is amino terminal to the putativetransmembrane domain, as defined by the ORF. These peptides were used tovalidate and provide reading frame information for the isolated clonesdescribed elsewhere in this application.

In additional studies carried out in support of the present invention,N-terminal sequencing of β-secretase isolated from additional cell typesrevealed that the N-terminus may be amino acid numbers 46, 22, 58, or 63with respect to the ORF sequence shown in FIG. 2A, depending on thetissue from which the protein is isolated, with the form having as itsN-terminus amino acid 46 predominating in the tissues tested. That is,in experiments carried out in support of the present invention, thefull-length β-secretase construct (i.e., encoding SEQ ID NO: 2) wastransfected into 293T cells and COS A2 cells, using the Fugene techniquedescribed in Example 6. β-secretase was isolated from the cells bypreparing a crude particulate fraction from the cell pellet, asdescribed in Example 5, followed by extraction with buffer containing0.2% Triton X-100. The Triton extract was diluted with pH 5.0 buffer andpassed through a SP Sepharose column, essentially according to themethods described in Example 5A. This step removed the majority ofcontaminating proteins. After adjusting the pH to 4.5, β-secretase wasfurther purified and concentrated on P10P4′staD→V Sepharose, asdescribed in Examples 5 and 7. Fractions were analyzed for N-terminalsequence, according to standard methods known in the art. Results aresummarized in Table 3, below.

The primary N-terminal sequence of the 293T cell-derived protein was thesame as that obtained from brain. In addition, minor amounts of proteinstarting just after the signal sequence (at Thr-22) and at the start ofthe aspartyl protease homology domain (Met-63) were also observed. Anadditional major form found in Cos A2 cells resulted from a Gly-58cleavage.

TABLE 3 N-terminal Sequences and Amounts of β-secretase Forms in VariousCell Types Est. Amount N-terminus Source (pmoles) (Ref.: SEQ ID NO: 2)Sequence Human brain 1–2 46 ETDEEPEEPGR (SEQ ID NO: 99) Recombinant,~35  46 ETDEEPEEPGR 293T ~7 22 (SEQ ID NO: 99) ~5 63 TQHGIRL(P)LR (SEQID NO: 100) MVDNLRGKS (SEQ ID NO: 101) Recombinant, ~4 46 ETDEEPEEPGRCosA2 ~3 58 (SEQ ID NO: 99) GSFVEMVDNL (SEQ ID NO: 102)

b. C-terminus

Further experiments carried out in support of the present inventionrevealed that the C-terminus of the full-length amino acid sequencepresented as SEQ ID NO: 2 can also be truncated, while still retainingβ-secretase activity of the molecule. More specifically, as described inmore detail in Part D below, C-terminal truncated forms of the enzymeending just before the putative transmembrane region, i.e. at or about10 amino acids C terminal to amino acid 452 with respect to SEQ ID NO:2, exhibit β-secretase activity, as evidenced by an ability to cleaveAPP at the appropriate cleavage site and/or ability to bind SEQ ID NO.72.

Thus, using the reference amino acid positions provided by SEQ ID NO: 2,one form of β-secretase extends from position 46 to position 501(β-secretase 46–501; SEQ ID NO: 43). Another form extends from position46 to any position including and beyond position 452, (β-secretase4–452+), with a preferred form being β-secretase 46-452 (SEQ ID NO: 58).More generally, another preferred form extends from position 1 to anyposition including and beyond position 452, but not including position501. Other active forms of the β-secretase protein begin at amino acid22, 58, or 63 and may extend to any point including and beyond thecysteine at position 420, and more preferably, including and beyondposition 452, while still retaining enzymatic activity (i.e.,β-secretase 22–452+; β-secretase 58–452+; β-secretase 63–452+). Asdescribed in Part D, below, those forms which are truncated at aC-terminal position at or before about position 452, or even severalamino acids thereafter, are particularly useful in crystallizationstudies, since they lack all or a significant portion of thetransmembrane region, which may interfere with protein crystallization.The recombinant protein extending from position 1 to 452 has beenaffinity purified using the procedures described herein.

C. Crystallization of β-secretase

According to a further aspect, the present invention also includespurified β-secretase in crystallized form, in the absence or presence ofbinding substrates, such as peptide, modified peptide, or small moleculeinhibitors. This section describes methods and utilities of suchcompositions.

1. Crystallization of the Protein

β-secretase purified as described above can be used as starting materialto determine a crystallographic structure and coordinates for theenzyme. Such structural determinations are particularly useful indefining the conformation and size of the substrate binding site. Thisinformation can be used in the design and modeling of substrateinhibitors of the enzyme. As discussed herein, such inhibitors arecandidate molecules for therapeutics for treatment of Alzheimer'sdisease and other amyloid diseases characterized by Aβ peptide amyloiddeposits.

The crystallographic structure of β-secretase is determined by firstcrystallizing the purified protein. Methods for crystallizing proteins,and particularly proteases, are now well known in the art. Thepractitioner is referred to Principles of Protein X-ray Crystallography(J. Drenth, Springer Verlag, N.Y., 1999) for general principles ofcrystallography. Additionally, kits for generating protein crystals aregenerally available from commercial providers, such as Hampton Research(Laguna Niguel, Calif.). Additional guidance can be obtained fromnumerous research articles that have been written in the area ofcrystallography of protease inhibitors, especially with respect to HIV-1and HIV-2 proteases, which are aspartic acid proteases.

Although any of the various forms of β-secretase described herein can beused for crystallization studies, particularly preferred forms lack thefirst 45 amino acids of the full length sequence shown as SEQ ID NO: 2,since this appears to be the predominant form which occurs naturally inhuman brain. It is thought that some form of post-translationalmodification, possibly autocatalysis, serves to remove the first 45amino acids in fairly rapid order, since, to date, virtually nonaturally occurring enzyme has been isolated with all of the first 45amino acids intact. In addition, it is considered preferable to removethe putative transmembrane region from the molecule prior tocrystallization, since this region is not necessary for catalysis andpotentially could render the molecule more difficult to crystallize.

Thus, a good candidate for crystallization is β-secretase 46–452 (SEQ IDNO: 58), since this is a form of the enzyme that (a) provides thepredominant naturally occurring N-terminus, and (b) lacks the “sticky”transmembrane region, while (c) retaining β-secretase activity.Alternatively, forms of the enzyme having extensions that extend part ofthe way (approximately 10–15 amino acids) into the transmembrane domainmay also be used. In general, for determining X-ray crystallographiccoordinates of the ligand binding site, any form of the enzyme can beused that either (i) exhibits β-secretase activity, and/or (ii) binds toa known inhibitor, such as the inhibitor ligand P 10-P4′staD→V, with abinding affinity that is at least 1/100 the binding affinity ofβ-secretase [46–501] (SEQ ID NO. 43) to P10-P4′staD→V (SEQ ID NO:72).Therefore, a number of additional truncated forms of the enzyme can beused in these studies. Suitability of any particular form can beassessed by contacting it with the P10-P4′staD→V affinity matrixdescribed above. Truncated forms of the enzyme that bind to the matrixare suitable for such further analysis. Thus, in addition to 46–452,discussed above, experiments in support of the present invention haverevealed that a truncated form ending in residue 419, most likely 46–419(SEQ ID NO:71), also binds to the affinity matrix and is therefore analternate candidate protein composition for X-ray crystallographicanalysis of β-secretase. More generally, any form of the enzyme thatends before the transmembrane domain, particularly those ending betweenabout residue 419 and 452 are suitable in this regard.

At the N-terminus, as described above, generally the first 45 aminoacids will be removed during cellular processing. Other suitablenaturally occurring or expressed forms are listed in Table 3 above.These include, for example, a protein commencing at residue 22, onecommencing at residue 58 and one commencing at residue 63. However,analysis of the entire enzyme, starting at residue 1, can also provideinformation about the enzyme. Other forms, such as 1–420 (SEQ ID NO 60)to 1–452 (SEQ ID NO: 59), including intermediate forms, for example1–440, can be useful in this regard. In general, it will also be usefulto obtain structure on any subdomain of the active enzyme.

Methods for purifying the protein, including active forms, are describedabove. In addition, since the protein is apparently glycosylated in itsnaturally occurring (and mammalian-expressed recombinant) forms, it maybe desirable to express the protein and purify it from bacterialsources, which do not glycosylate mammalian proteins, or express it insources, such as insect cells, that provide uniform glycosylationpatterns, in order to obtain a homogeneous composition. Appropriatevectors and codon optimization procedures for accomplishing this areknown in the art.

Following expression and purification, the protein is adjusted to aconcentration of about 1–20 mg/ml. In accordance with methods that haveworked for other crystallized proteins, the buffer and saltconcentrations present in the initial protein solution are reduced to aslow a level as possible. This can be accomplished by dialyzing thesample against the starting buffer, using microdialysis techniques knownin the art. Buffers and crystallization conditions will vary fromprotein to protein, and possibly from fragment to fragment of the activeβ-secretase molecule, but can be determined empirically using, forexample, matrix methods for determining optimal crystallizationconditions. (Drentz, J., supra; Ducruix, A., et al., eds.Crystallization of Nucleic Acids and Proteins: A Practical Approach,Oxford University Press, New York, 1992.)

Following dialysis, conditions are optimized for crystallization of theprotein. Generally, methods for optimization may include making a “grid”of 1 μl drops of the protein solution, mixed with 1 μl well solution,which is a buffer of varying pH and ionic strength. These drops areplaced in individual sealed wells, typically in a “hanging drop”configuration, for example in commercially available containers (HamptonResearch, Laguna Niguel, CA). Precipitation/crystallization typicallyoccurs between 2 days and 2 weeks. Wells are checked for evidence ofprecipitation or crystallization, and conditions are optimized to formcrystals. Optimized crystals are not judged by size or morphology, butrather by the diffraction quality of crystals, which should providebetter than 3 Å resolution. Typical precipitating agents includeammonium sulfate (NH₄SO₄), polyethylene glycol (PEG) and methyl pentanediol (MPD). All chemicals used should be the highest grade possible(e.g., ACS) and may also be re-purified by standard methods known in theart, prior to use.

Exemplary buffers and precipitants forming an empirical grid fordetermining crystallization conditions are commercially available. Forexample, the “Crystal Screen” kit (Hampton Research) provides a sparsematrix method of trial conditions that is biased and selected from knowncrystallization conditions for macromolecules. This provides a “grid”for quickly testing wide ranges of pH, salts, and precipitants using avery small sample (50 to 100 microliters) of macromolecule. In suchstudies, 1 μl of buffer/precipitant(s) solution is added to an equalvolume of dialyzed protein solution, and the mixtures are allowed to sitfor at least two days to two weeks, with careful monitoring ofcrystallization. Chemicals can be obtained from common commercialsuppliers; however, it is preferable to use purity grades suitable forcrystallization studies, such as are supplied by Hampton Research(Laguna Niguel, CA). Common buffers include Citrate, TEA, CHES, Acetate,ADA and the like (to provide a range of pH optima), typically at aconcentration of about 100 mM. Typical precipitants include (NH₄)₂SO₄,MgSO₄, NaCl, MPD, Ethanol, polyethylene glycol of various sizes,isopropanol, KCl; and the like (Ducruix).

Various additives can be used to aid in improving the character of thecrystals, including substrate analogs, ligands, or inhibitors, asdiscussed in Part 2, below, as well as certain additives, including, butnot limited to:

5% Jeffamine

5% Polypropyleneglycol P400

5% Polyethyleneglycol 400

5% ethyleneglycol

5% 2-methyl-2,4-pentanediol

5% Glycerol

5% Dioxane

5% dimethyl sulfoxide

5% n-Octanol

100 mM (NH₄)₂SO₄

100 mM CsCl

100 mM CoSO4

100 mM MnCl2

100 mM KCl

100 mM ZnSO4

100 mM LiCl2

100 mM MgCl₂

100 mM Glucose

100 mM 1,6-Hexanediol 100 mM Dextran sulfate

100 mM 6-amino caproic acid

100 mM 1,6 hexane diamine

100 mM 1,8 diamino octane

100 mM Spermidine

100 mM Spermine

0.17 mM n-dodecyl-β-D-maltoside NP 40

20 mM n-octyl-β-D-glucopyranoside

According to one discovery of the present invention, the full-lengthβ-secretase enzyme contains at least one transmembrane domain, and itspurification is aided by the use of a detergent (Triton X-100). Membraneproteins can be crystallized intact, but may require specializedconditions, such as the addition of a non-ionic detergent, such as C₈G(8-alkyl-β-glucoside) or an n-alkyl-maltoside (C_(n)M). Selection ofsuch a detergent is somewhat empirical, but certain detergents arecommonly employed. A number of membrane proteins have been successfully“salted out” by addition of high salt concentrations to the mixture. PEGhas also been used successfully to precipitate a number of membraneproteins (Ducruix, et al., supra). Alternatively, as discussed above, aC-terminal truncated form of the protein that binds inhibitor but whichlacks the transmembrane domain, such as β-secretase 46–452 (SEQ IDNO:58), is crystallized.

After crystallization conditions are determined, crystallization of alarger amount of the protein can be achieved by methods known in theart, such as vapor diffusion or equilibrium dialysis. In vapordiffusion, a drop of protein solution is equilibrated against a largerreservoir of solution containing precipitant or another dehydratingagent. After sealing, the solution equilibrates to achievesupersaturating concentrations of proteins and thereby inducecrystallization in the drop.

Equilibrium dialysis can be used for crystallization of proteins at lowionic strength. Under these conditions, a phenomenon known as “saltingin” occurs, whereby the protein molecules achieve balance ofelectrostatic charges through interactions with other protein molecules.This method is particularly effective when the solubility of the proteinis low at the lower ionic strength. Various apparatuses and methods areused, including microdiffusion cells in which a dialysis membrane isattached to the bottom of a capillary tube, which may be bent at itslower portion. The final crystallization condition is achieved by slowlychanging the composition of the outer solution. A variation of thesemethods utilizes a concentration gradient equilibrium dialysis set up.Microdiffusion cells are available from commercial suppliers such asHampton Research (Laguna Niguel, Calif.).

Once crystallization is achieved, crystals characterized for purity(e.g., SDS-PAGE) and biological activity. Larger crystals (>0.2 mm) arepreferred to increase the resolution of the X-ray diffraction, which ispreferably on the order of 10–1.5 Angstroms. The selected crystals aresubjected to X-ray diffraction, using a strong, monochromatic X-raysource, such as a Synchrotron source or rotating anode generator, andthe resulting X-ray diffraction patterns are analyzed, using methodsknown in the art.

In one application, β-secretase amino acid sequence and/or X-raydiffraction data is recorded on computer readable medium, by which ismeant any medium that can be read and directly acccessed by a computer.These data may be used to model the enzyme, a subdomain thereof, or aligand thereof. Computer algorithms useful for this application arepublicly and commercially available.

2. Crystallization of Protein plus Inhibitor

As mentioned above, it is advantageous to co-crystallize the protein inthe presence of a binding ligand, such as inhibitor. Generally, theprocess for optimizing crystallization of the protein is followed, withaddition of greater than 1 mM concentration of the inhibitor ligandduring the precipitation phase. These crystals are also compared tocrystals formed in the absence of ligand, so that measurements of theligand binding site can be made. Alternatively, 1–2 μl of 0.1–25 mMinhibitor compound is added to the drop containing crystals grown in theabsence of inhibitor in a process known as “soaking.” Based on thecoordinates of the binding site, further inhibitor optimization isachieved. Such methods have been used advantageously in finding new,more potent inhibitors for HIV proteases (See, e.g., Viswanadhan, V. N.,et al J. Med. Chem. 39: 705–712, 1996; Muegge, J., et al. J. Med. Chem.42: 791–804, 1999).

One inhibitor ligand which is used in these co-crystallization andsoaking experiments is P10-P4′staD→V (SEQ ID NO: 72), a statin peptideinhibitor described above. Methods for making the molecule are describedherein. The inhibitor is mixed with β-secretase, and the mixture issubjected to the same optimization tests described above, concentratingon those conditions worked out for the enzyme alone. Coordinates aredetermined and comparisons are made between the free and ligand boundenzyme, according to methods well known in the art. Further comparisonscan be made by comparing the inhibitory concentrations of the enzyme tosuch coordinates, such as described by Viswanadhan, et al, supra.Analysis of such comparisons provides guidance for design of furtherinhibitors, using this method.

D. Biological Activity of β-secretase

1. Naturally occurring β-secretase

In studies carried out in support of the present invention, isolated,purified forms of β-secretase were tested for enzymatic activity usingone or more native or synthetic substrates. For example, as discussedabove, when β-secretase was prepared from human brain and purified tohomogeneity using the methods described in Example 5A, a single band wasobserved by silver stain after electrophoresis of sample fractions fromthe anion exchange chromatography (last step) on an SDS-polyacrylamidegel under reducing (+β-mercaptoethanol) conditions. As summarized inTable 1, above, this fraction yielded a specific activity ofapproximately 1.5×10⁹ nM/h/mg protein, where activity was measured byhydrolysis of MBP-C125SW.

2. Isolated Recombinant β-secretase

Various recombinant forms of the enzyme were produced and purified fromtransfected cells. Since these cells were made to overproduce theenzyme, it was found that the purification scheme described with respectnaturally occurring forms of the enzyme (e.g., Example 5A) could beshortened, with positive results. For example, as detailed in Example6,293T cells were transfected with pCEK clone 27 (FIG. 12 and FIGS.13A–E) (SEQ ID NO:48 and Cos A2 cells were transfected with pCFβA2 using“FUGENE” 6 Transfection Reagent (Roche Molecular Biochemicals Research,Indianapolis, Ind.). The vector pCF was constructed from the parentvector pcDNA3, commercially available from Invitrogen, by inserting SEQID NO: 80 (FIG. 11A) between the HindIII and EcoRI sites. This sequenceencompasses the adenovirus major late promoter tripartite leadersequence, and a hybrid splice created from adenovirus major late regionfirst exon and intron and a synthetically generated IgG variable regionsplice acceptor.

pcDNA3 was cut with restriction endonucleases HindIII and EcoRI, thenblunted by filling in the ends with Klenow fragment of DNA polymerase I.The cut and blunted vector was gel purified, and ligated with isolatedfragment from pED.GI. The pED fragment was prepared by digesting withPvuII and SmaI, followed by gel purification of the resulting 419base-pair fragment, which was further screened for orientation, andconfirmed by sequencing.

To create the pCEK expression vector, the expression cassette from pCFwas transferred into the EBV expression vector pCEP4 (Invitrogen,Carlsbad, Calif.). pCEP 4 was cut with BglII and XbaI, filled in, andthe large 9:15 kb fragment containing pBR, hygromycin, and EBVsequences) ligated to the 1.9 kb NruI to XmnI fragment of pCF containingthe expression cassette (CMV, TPL/MLP/IGg splice, Sp6, SVpolyA, M13flanking region). pCFβA2 (clone A2) contains full length β-secretase inthe vector pCF. pCF vector replicates in COS and 293T cells. In eachcase, cells were pelleted and a crude particulate fraction was preparedfrom the pellet. This fraction was extracted with buffer containing 0.2%Triton X-100. The Triton extract was diluted with pH 5.0 buffer andpassed through a SP Sepharose column. After the pH was adjusted to 4.5,β-secretase activity containing fractions were concentrated, with someadditional purification on P10-P4′(statine)D→V Sepharose, as describedfor the brain enzyme. Silver staining of fractions revealed co-purifiedbands on the gel. Fractions corresponding to these bands were subjectedto N-terminal amino acid determination. Results from these experimentsrevealed some heterogeneity of β-secretase species within the fractions.These species represent various forms of the enzyme; for example, fromthe 293T cells, the primary N-terminus is the same as that found in thebrain, where (with respect to SEQ ID NO: 2) amino acid 46 is at theN-terminus. Minor amounts of protein starting just after the signalsequence (at residue 23) and at the start of the aspartyl proteasehomology domain (Met-63) were also observed. An additional major form ofprotein was found in Cos A2 cells, resulting from cleavage at Gly-58.These results are summarized in Table 3, above.

2. Comparison of Isolated, Naturally Occurring β-secretase withRecombinant

β-secretase

As described above, naturally occurring β-secretase derived from humanbrain as well as recombinant forms of the enzyme exhibit activity incleaving APP, particularly as evidenced by activity in the MBP-C125assay. Further, key peptide sequences from the naturally occurring formof the enzyme match portions of the deduced sequence derived fromcloning the enzyme. Further confirmation that the two enzymes actidentically can be taken from additional experiments in which variousinhibitors were found to have very similar affinities for each enzyme,as estimated by a comparison of IC₅₀ values measured for each enzymeunder similar assay conditions. These inhibitors were discovered inaccordance with a further aspect of the invention, which is describedbelow. Significantly, the inhibitors produce near identical IC₅₀ valuesand rank orders of potency in brain-derived and recombinant enzymepreparations, when compared in the same assay.

In further studies, comparisons were made between the full lengthrecombinant enzyme having a C-terminal flag sequence “FLp501” (SEQ IDNO: 2, +SEQ ID NO: 45) and a recombinant enzyme truncated at position452 “452Stop” (SEQ ID NO: 58 or SEQ ID NO: 59). Both enzymes exhibitedactivity in cleaving β-secretase substrates such as MBP-C125, asdescribed above. The C-terminal truncated form of the enzyme exhibitedactivity in cleaving the MBP-C125sw substrate as well as the P26-P4′substrate, with similar rank order of potency for the various inhibitordrugs tested. In addition, the absolute IC₅₀s were comparable for thetwo enzymes tested with the same inhibitor. All IC₅₀s were less than 10μM.

1. Cellular β-secretase

Further experiments carried out in support of the present invention haverevealed that the isolated β-secretase polynucleotide sequencesdescribed herein encode β-secretase or α-secretase fragments that areactive in cells. This section describes experiments carried out insupport of the present invention, cells were transfected with DNAencoding β-secretase alone, or were co-transfected with DNAencoding-secretase and DNA encoding wild-type APP as detailed in Example8.

a. Transfection with β-secretase

In experiments carried out in support of the present invention, clonescontaining genes expressing the full-length polypeptide (SEQ ID NO: 2)were transfected into COS cells (Fugene and Effectene methods). Wholecell lysates were prepared and various amounts of lysate were tested forβ-secretase activity according to standard methods known in the art ordescribed in Example 4 herein. FIG. 14B shows the results of theseexperiments. As shown, lysates prepared from transfected cells, but notfrom mock- or control cells, exhibited considerable enzymatic activityin the MPB-C125sw assay, indicating “overexpression” of β-secretase bythese cells.

b. Co-transfection of Cells with β-secretase and APP

In further experiments, 293T cells were co-transfected with pCEK clone27, FIGS. 12 and 13 or poCK vector containing the full lengthβ-secretase molecule (1–501; SEQ ID NO: 2) and with a plasmid containingeither the wild-type or Swedish APP construct pohCK751, as described inExample 8. β-specific cleavage was analyzed by ELISA and Westernanalyses to confirm that the correct site of cleavage occurs.

Briefly, 293T cells were co-transfected with equivalent amounts ofplasmids encoding βAPPsw or wt and β-secretase or controlβ-galactosidase (β-gal) cDNA according to standard methods. βAPP andβ-secretase cDNAs were delivered via vectors, pohCK or pCEK, which donot replicate in 293T cells (pCEK-clone 27, FIGS. 12 and 13; pohCK751expressing βAPP 751, FIG. 21). Conditioned media and cell lysates werecollected 48 hours after transfection. Western assays were carried outon conditioned media and cell lysates. ELISAs for detection of Aβpeptide were carried out on the conditioned media to analyze various APPcleavage products.

Western Blot Results

It is known that β-secretase specifically cleaves at the Met-Asp inAPPwt and the Leu-Asp in APPsw to produce the Aβ peptide, starting atposition 1 and releasing soluble APP (sAPPβ). Immunological reagents,specifically antibody 92 and 92sw (or 192sw), respectively, have beendeveloped that specifically detect cleavage at this position in theAPPwt and APPsw substrates, as described in U.S. Pat. No. 5,721,130,incorporated herein by reference. Western blot assays were carried outon gels on which cell lysates were separated. These assays wereperformed using methods well known in the art, using as primary antibodyreagents Ab 92 or Ab92S, which are specific for the C terminus of theN-terminal fragment of APP derived from APPwt and APPsw, respectively.In addition, ELISA format assays were performed using antibodiesspecific to the N terminal amino acid of the C terminal fragment.

Monoclonal antibody 13G8 (specific for C-terminus of APP—epitope atpositions 675–695 of APP695) was used in a Western blot format todetermine whether the transfected cells express APP. FIG. 15A shows thatreproducible transfection was obtained with expression levels of APP invast excess over endogenous levels (triplicate wells are indicated as 1,2, 3 in FIG. 15A). Three forms of APP—mature, immature andendogenous—can be seen in cells transfected with APPwt or APPsw. Whenβ-secretase was co-transfected with APP, smaller C-terminal fragmentsappeared in triplicate well lanes from co-transfected cells (Westernblot FIG. 15A, right-most set of lanes). In parallel experiments, wherecells were co-transfected with β-secretase and APPsw substrate,literally all of the mature APP was cleaved (right-most set of laneslabeled “1,2,3” of FIG. 15B). This suggests that there is extensivecleavage by β-secretase of the mature APP (upper band), which results inC-terminal fragments of expected size in the lysate for cleavage at theβ-secretase site. Co-transfection with Swedish substrate also resultedin an increase in two different sized CTF fragments (indicated by star).In conjuction with the additional Western and ELISA results describedbelow, these results are consistent with a second cleavage occurring onthe APPsw substrate after the initial cleavage at the β-secretase site.

Conditioned medium from the cells was analyzed for reactivity with the192sw antibody, which is specific for β-s-APPsw. Analysis using thisantibody indicated a dramatic increase in β-secretase cleaved solubleAPP. This is observed in the gel illustrated in FIG. 16B by comparingthe dark bands present in the “APPsw βsec” samples to the bands presentin the “APPsw βgal” samples. Antibody specific for β-s-APPwt alsoindicates an increase in β-secretase cleaved material, as illustrated inFIG. 16A.

Since the antibodies used in these experiments are specific for theβ-secretase cleavage site, the foregoing results show that p501β-secretase cleaves APP at this site, and the overexpression of thisrecombinant clone leads to a dramatic enhancement of β-secretaseprocessing at the correct β-secretase site in whole cells. Thisprocessing works on the wildtype APP substrate and is enhancedsubstantially on the Swedish APP substrate. Since approximately 20% ofsecreted APP in 293T cells is β-sAPP, with the increase observed belowfor APPsw, it is probable that almost all of the sAPP is β-sAPP. Thisobservation was further confirmed by independent Western assays in whichalpha and total sAPP were measured.

Monoclonal antibody 1736 is specific for the exposed α-secretase cleavedβ-APP (Selkoe, et al.). When Western blots were performed using thisantibody as primary antibody, a slight but reproducible decrease inα-cleaved APPwt was observed (FIG. 17A), and a dramatic decrease inα-cleaved APPsw material was also observed (note near absence ofreactivity in FIG. 17B in the lanes labeled “APPsw βsec”). These resultssuggest that the overexpressed recombinant p501 β-secretase cleavesAPPsw so efficiently or extensively that there is little or no substrateremaining for α-secretase to cleave. This further indicates that all thesAPP in APPsw βsec samples (illustrated in FIG. 16B) is β-sAPP.

Aβ ELISA Results

Conditioned media from the recombinant cells was collected, diluted asnecessary and tested for Aβ peptide production by ELISA on microtiterplates coated with monoclonal antibody 2G3, which is specific forrecognizing the C-terminus of Aβ(x-40), with the detector reagentbiotinylated mAb 3D6, which measures Aβ(x-40) (i.e., allN-terminus-truncated forms of the Aβ peptide). Overexpression ofβ-secretase with APPwt resulted in an approximately 8-fold increase inAβ(x-40) production, with 1–40 representing a small percentage of thetotal. There was also a substantial increase in the production of Aβ1–40 (FIG. 18). With APPsw there was an approximate 2-fold increase inAβ(x-40). Without adhering to any particular underlying theory, it isthought that the less dramatic increase of Aβ(x-40) β-sec/APPsw cells incomparison to the β-sec/APPwt cells is due in part to the fact thatprocessing of the APPsw substrate is much more efficient than that ofthe APPwt substrate. That is, a significant amount of APPsw is processedby endogenous β-secretase, so further increases upon transfection ofβ-secretase are therefore limited. These data indicate that theexpression of recombinant β-secretase increases Aβ production and thatβ-secretase is rate limiting for production of Aβ in cells. This meansthat β-secretase enzymatic activity is rate limiting for production ofAβ in cells, and therefore provides a good therapeutic target.

IV. Utility

A. Expression Vectors and Cells Expressing β-secretase

The invention includes further cloning and expression of members of theaspartyl protease family described above, for example, by insertingpolynucleotides encoding the proteins into standard expression vectorsand transfecting appropriate host cells according to standard methodsdiscussed below. Such expression vectors and cells expressing, forexample, the human β-secretase enzyme described herein, have utility,for example, in producing components (purified enzyme or transfectedcells) for the screening assays discussed in Part B, below. Suchpurified enzyme also has utility in providing starting materials forcrystallization of the enzyme, as described in Section III, above. Inparticular, truncated form(s) of the enzyme, such as 1–452 (SEQ IDNO:59) and 46–452 (SEQ ID NO:58), and the deglycosylated forms of theenzyme described herein are considered to have utility in this regard,as are other forms truncated partway into the transmembrane region, forexample amino acid residues 1–460 or 46–458, respectively, in referenceto SEQ ID NO:2.

In accordance with the present invention, polynucleotide sequences whichencode human β-secretase, splice variants, fragments of the protein,fusion proteins, or functional equivalents thereof, collectivelyreferred to herein as “β-secretase,” may be used in recombinant DNAmolecules that direct the expression of β-secretase in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other nucleicacid sequences that encode substantially the same or a functionallyequivalent amino acid sequence may be used to clone and expressβ-secretase. Such variations will be readily ascertainable to personsskilled in the art.

The polynucleotide sequences of the present invention can be engineeredin order to alter a β-secretase coding sequence for a variety ofreasons, including but not limited to, alterations that modify thecloning, processing and/or expression of the gene product. For example,alterations may be introduced using techniques which are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, to alter glycosylation patterns, to change codon preference, toproduce splice variants, etc. For example, it may be advantageous toproduce β-secretase-encoding nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particularprokaryotic or eukaryotic host (Murray, E. et al. (1989) Nuc Acids Res17:477–508) can be selected, for example, to increase the rate ofβ-secretase polypeptide expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence. This may beparticularly useful in producing recombinant enzyme in non-mammaliancells, such as bacterial, yeast, or insect cells. The present inventionalso includes recombinant constructs comprising one or more of thesequences as broadly described above. The constructs comprise a vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. In apreferred aspect of this embodiment, the construct further comprisesregulatory sequences, including, for example, a promoter, operablylinked to the sequence. Large numbers of suitable vectors and promotersare known to those of skill in the art, and are commercially available.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are also described in Sambrook, et al., (supra).

The present invention also relates to host cells that are geneticallyengineered with vectors of the invention, and the production of proteinsand polypeptides of the invention by recombinant techniques. Host cellsare genetically engineered (i.e., transduced, transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the β-secretase gene. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. Exemplary methods for transfection of varioustypes of cells are provided in Example 6, herein.

As described above, according to a preferred embodiment of theinvention, host cells can be co-transfected with an enzyme substrate,such as with APP (such as wild type or Swedish mutation form), in orderto measure activity in a cell environment. Such host cells are ofparticular utility in the screening assays of the present invention,particularly for screening for therapeutic agents that are able totraverse cell membranes.

The polynucleotides of the present invention may be included in any of avariety of expression vectors suitable for expressing a polypeptide.Such vectors include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. However, any other vector may be used as longas it is replicable and viable in the host. The appropriate DNA sequencemay be inserted into the vector by a variety of procedures. In general,the DNA sequence is inserted into an appropriate restrictionendonuclease site(s) by procedures known in the art. Such procedures andrelated sub-cloning procedures are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate transcription control sequence (promoter) to direct mRNAsynthesis. Examples of such promoters include: CMV, LTR or SV40promoter, the E. coli lac or trp promoter, the phage lambda PL promoter,and other promoters known to control expression of genes in prokaryoticor eukaryotic cells or their viruses. The expression vector alsocontains a ribosome binding site for translation initiation, and atranscription terminator. The vector may also include appropriatesequences for amplifying expression. In addition, the expression vectorspreferably contain one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as described above,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express theprotein. Examples of appropriate expression hosts include: bacterialcells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungalcells, such as yeast; insect cells such as Drosophila and SpodopteraSf9; mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma;adenoviruses; plant cells, etc. It is understood that not all cells orcell lines will be capable of producing fully functional β-secretase;for example, it is probable that human β-secretase is highlyglycosylated in native form, and such glycosylation may be necessary foractivity. In this event, eukaryotic host cells may be preferred. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein. The invention is notlimited by the host cells employed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for β-secretase. For example, when largequantities of β-secretase or fragments thereof are needed for theinduction of antibodies, vectors, which direct high level expression offusion proteins that are readily purified, may be desirable. Suchvectors include, but are not limited to, multifunctional E. coli cloningand expression vectors such as Bluescript(R) (Stratagene, La Jolla,Calif.), in which the β-secretase coding sequence may be ligated intothe vector in-frame with sequences for the amino-terminal Met and thesubsequent 7 residues of beta-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem264:5503–5509); pET vectors (Novagen, Madison Wis.); and the like.

In the yeast Saccharomyces cerevisiae a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al. (supra) andGrant et al. (1987; Methods in Enzymology 153:516–544).

In cases where plant expression vectors are used, the expression of asequence encoding β-secretase may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV (Brisson et al. (1984) Nature 310:511–514) may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu et al. (1987) EMBO J. 6:307–311). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al (1984)EMBO J. 3:1671–1680; Broglie et al. (1984) Science 224:838–843); or heatshock promoters (Winter J and Sinibaldi R M (1991) Results. Probi. CellDiffer. 17:85–105) may be used. These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. For reviews of such techniques, see Hobbs S or Murry L E(1992) in McGraw Hill Yearbook of Science and Technology, McGraw Hill,New York, N.Y., pp 191–196; or Weissbach and Weissbach (1988) Methodsfor Plant Molecular Biology, Academic Press, New York, N.Y., pp 421–463.

β-secretase may also be expressed in an insect system. In one suchsystem, Autographa californica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes in Spodoptera frugiperda Sf9cells or in Trichoplusia larvae. The β-secretase coding sequence iscloned into a nonessential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of Kv-SL coding sequence will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein coat. Therecombinant viruses are then used to infect S. frugiperda cells orTrichoplusia larvae in which β-secretase is expressed (Smith et al.(1983) J Virol 46:584; Engelhard E K et al. (1994) Proc Nat Acad Sci91:3224–3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a β-secretase coding sequence may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingthe enzyme in infected host cells (Logan and Shenk (1984) Proc Natl AcadSci 81:3655–3659). In addition, transcription enhancers, such as therous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be required for efficienttranslation of a α-secretase coding sequence. These signals include theATG initiation codon and adjacent sequences. In cases where β-secretasecoding sequence, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate to the cell system inuse (Scharf D et al. (1994) Results Probi Cell Differ 20:125–62; Bittneret al. (1987) Methods in Enzymol 153:516–544).

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., and Battey, 1.(1986) Basic Methods in Molecular Biology) or newer methods, includinglipid transfection with “FUGENE” (Roche Molecular Biochemicals,Indianapolis, Ind.) or “EFFECTENE” (Quiagen, Valencia, Calif.), or otherDNA carrier molecules. Cell-free translation systems can also beemployed to produce polypeptides using RNAs derived from the DNAconstructs of the present invention.

A host cell strain may be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingwhich cleaves a “prepro” form of the protein may also be important forcorrect insertion, folding and/or function. For example, in the case ofβ-secretase, it is likely that the N-terminus of SEQ ID NO: 2 istruncated, so that the protein begins at amino acid 22, 46 or 57–58 ofSEQ ID NO: 2. Different host cells such as CHO, HeLa, BHK, MDCK, 293,W138, etc. have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the introduced,foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression may be preferred. For example, cell lines that stably expressβ-secretase may be transformed using expression vectors which containviral origins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1–2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type. For example, inexperiments carried out in support of the present invention,overexpression of the “452stop” form of the enzyme has been achieved.

Host cells transformed with a nucleotide sequence encoding β-secretasemay be cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture. The protein orfragment thereof produced by a recombinant cell may be secreted,membrane-bound, or contained intracellularly, depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides encoding β-secretasecan be designed with signal sequences which direct secretion ofβ-secretase polypeptide through a prokaryotic or eukaryotic cellmembrane.

β-secretase may also be expressed as a recombinant protein with one ormore additional polypeptide domains added to facilitate proteinpurification. Such purification facilitating domains include, but arenot limited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle, Wash.). The inclusion of a protease-cleavable polypeptidelinker sequence between the purification domain and β-secretase isuseful to facilitate purification. One such expression vector providesfor expression of a fusion protein comprising β-secretase (e.g., asoluble β-secretase fragment) fused to a polyhistidine region separatedby an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography, asdescribed in Porath et al. (1992) Protein Expression and Purification3:263–281) while the enterokinase cleavage site provides a means forisolating β-secretase from the fusion protein. pGEX vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, orother methods, which are well know to those skilled in the art.

β-secretase can be recovered and purified from recombinant cell culturesby any of a number of methods well known in the art, or, preferably, bythe purification scheme described herein. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Details of methods for purifying naturally occurring as well as purifiedforms of β-secretase are provided in the Examples.

B. Methods of Selecting β-secretase Inhibitors

The present invention also includes methods for identifying molecules,such as synthetic drugs, antibodies, peptides, or other molecules, whichhave an inhibitory effect on the activity of β-secretase describedherein, generally referred to as inhibitors, antagonists or blockers ofthe enzyme. Such an assay includes the steps of providing a humanβ-secretase, such as the β-secretase which comprises SEQ ID NO: 2, SEQID NO: 43, or more particularly in reference to the present invention,an isolated protein, about 450 amino acid residues in length, whichincludes an amino acid sequence that is at least 90% identical to SEQ IDNO: 75 [63–423] including conservative substitutions thereof, whichexhibits α-secretase activity, as described herein. The β-secretaseenzyme is contacted with a test compound to determine whether it has amodulating effect on the activity of the enzyme, as discussed below, andselecting from test compounds capable of modulating β-secretaseactivity. In particular, inhibitory compounds (antagonists) are usefulin the treatment of disease conditions associated with amyloiddeposition, particularly Alzheimer's disease. Persons skilled in the artwill understand that such assays may be conveniently transformed intokits.

Particularly useful screening assays employ cells which express bothβ-secretase and APP. Such cells can be made recombinantly byco-transfection of the cells with polynucleotides encoding the proteins,as described in Section III, above, or can be made by transfecting acell which naturally contains one of the proteins with the secondprotein. In a particular embodiment, such cells are grown up inmulli-well culture dishes and are exposed to varying concentrations of atest compound or compounds for a pre-determined period of time, whichcan be determined empirically. Whole cell lysates, cultured media orcell membranes are assayed for β-secretase activity. Test compoundswhich significantly inhibit activity compared to control (as discussedbelow) are considered therapeutic candidates.

Isolated β-secretase, its ligand-binding, catalytic, or immunogenicfragments, or oligopeptides thereof, can be used for screeningtherapeutic compounds in any of a variety of drug screening techniques.The protein employed in such a test may be membrane-bound, free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenβ-secretase and the agent being tested can be measured. Compounds thatinhibit binding between β-secretase and its substrates, such as APP orAPP fragments, may be detected in such an assay. Preferably, enzymaticactivity will be monitored, and candidate compounds will be selected onthe basis of ability to inhibit such activity. More specifically, a testcompound will be considered as an inhibitor of β-secretase if themeasured β-secretase activity is significantly lower than β-secretaseactivity measured in the absence of test compound. In this context, theterm “significantly lower” means that in the presence of the testcompound the enzyme displays an enzymatic activity which, when comparedto enzymatic activity measured in the absence of test compound, ismeasurably lower, within the confidence limits of the assay method. Suchmeasurements can be assessed by a change in K_(m) and/or V_(max), singleassay endpoint analysis, or any other method standard in the art.Exemplary methods for assaying β-secretase are provided in Example 4herein.

For example, in studies carried out in support of the present invention,compounds were selected based on their ability to inhibit β-secretaseactivity in the MBP-C125 assay. Compounds that inhibited the enzymeactivity at a concentration lower than about 50 μM were selected forfurther screening.

The groups of compounds that are most likely candidates for inhibitoractivity comprise a further aspect of the present invention. Based onstudies carried out in support of the invention, it has been determinedthat the peptide compound described herein as P10-P4′staD→V (SEQ ID NO:72) is a reasonably potent inhibitor of the enzyme. Further studiesbased on this sequence and peptidomimetics of portions of this sequencehave revealed a number of small molecule inhibitors.

Random libraries of peptides or other compounds can also be screened forsuitability as β-secretase inhibitors. Combinatorial libraries can beproduced for many types of compounds that can be synthesized in astep-by-step fashion. Such compounds include polypeptides, beta-turnmimetics, polysaccharides, phospholipids, hormones, prostaglandins,steroids, aromatic compounds, heterocyclic compounds, benzodiazepines,oligomeric N-substituted glycines and oligocarbamatesi. Largecombinatorial libraries of the compounds can be constructed by theencoded synthetic libraries (ESL) method described in Affymax, WO95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051,Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which isincorporated by reference for all purposes).

A preferred source of test compounds for use in screening fortherapeutics or therapeutic leads is a phage display library. See, e.g.,Devlin, WO 91/18980; Key, B. K., et al, eds., Phage Display of Peptidesand Proteins, A Laboratory Manual, Academic Press, San Diego, CA, 1996.Phage display is a powerful technology that allows one to use phagegenetics to select and amplify peptides or proteins of desiredcharacteristics from libraries containing 10⁸–10⁹ different sequences.Libraries can be designed for selected variegation of an amino acidsequence at desired positions, allowing bias of the library towarddesired characteristics. Libraries are designed so that peptides areexpressed fused to proteins that are displayed on the surface of thebacteriophage. The phage displaying peptides of the desiredcharacteristics are selected and can be regrown for expansion. Since thepeptides are amplified by propagation of the phage, the DNA from theselected phage can be readily sequenced facilitating rapid analyses ofthe selected peptides.

Phage encoding peptide inhibitors can be selected by selecting for phagethat bind specifically to β-secretase protein. Libraries are generatedfused to proteins such as gene II that are expressed on the surface ofthe phage. The libraries can be composed of peptides of various lengths,linear or constrained by the inclusion of two Cys amino acids, fused tothe phage protein or may also be fused to additional proteins as ascaffold. One may start with libraries composed of random amino acids orwith libraries that are biased to sequences in the βAPP substratesurrounding the β-secretase cleavage site or preferably, to the D→Vsubstituted site exemplified in SEQ ID NO: 72. One may also designlibraries biased toward the peptidic inhibitors and substrates describedherein or biased toward peptide sequences obtained from the selection ofbinding phage from the initial libraries provide additional testinhibitor compound.

The β-secretase is immobilized and phage specifically binding to theβ-secretase selected for. Limitations, such as a requirement that thephage not bind in the presence of a known active site inhibitor ofβ-secretase (e.g. the inhibitors described herein), serve to furtherdirect phage selection active site specific compounds. This can becomplicated by a differential selection format. Highly purifiedβ-secretase, derived from brain or preferably from recombinant cells canbe immobilized to 96 well plastic dishes using standard techniques(reference phage book). Recombinant β-secretase, designed to be fused toa peptide that can bind (e.g. strepaviden binding motifs, His, FLAG ormyc tags) to another protein immobilized (such as streptavidin orappropriate antibodies) on the plastic petri dishes can also be used.Phage are incubated with the bound β-secretase and unbound phage removedby washing. The phage are eluted and this selection is repeated until apopulation of phage binding to β-secretase is recovered. Binding andelution are carried out using standard techniques.

Alternatively β-secretase can be “bound” by expressing it in Cos orother mammalian cells growing on a petri dish. In this case one wouldselect for phage binding to the α-secretase expressing cells, and selectagainst phage that bind to the control cells, that are not expressingβ-secretase.

One can also use phage display technology to select for preferredsubstrates of β-secretase, and incorporate the identified features ofthe preferred substrate peptides obtained by phage display intoinhibitors.

In the case of β-secretase, knowledge of the amino acid sequencesurrounding the cleavage site of APP and of the cleavage site of APPswhas provided information for purposes of setting up the phage displayscreening library to identify preferred substrates of β-secretase. Asmentioned above, knowledge of the sequence of a particularly goodpeptide inhibitor, P10P4staD→V (SEQ ID NO:72, as described herein,provides information for setting up a “biased” library toward thissequence.

For example, the peptide substrate library containing 10⁸ differentsequences is fused to a protein (such as a gene III protein) expressedon the surface of the phage and a sequence that can be used for bindingto streptavidin, or another protein, such as His tag and antibody toHis. The phage are digested with protease, and undigested phage areremoved by binding to appropriate immobilized binding protein, such asstreptavidin. This selection is repeated until a population of phageencoding substrate peptide sequences is recovered. The DNA in the phageis sequenced to yield the substrate sequences. These substrates are thenused for further development of peptidomimetics, particularlypeptidomimetics having inhibitory properties.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to, or preferably, toinhibit β-secretase activity in any of the assays described herein orotherwise known in the art. Compounds identified by such screens arethen further analyzed for potency in such assays. Inhibitor compoundscan then be tested for prophylactic and therapeutic efficacy intransgenic animals predisposed to an amyloidogenic disease, such asvarious rodents bearing a human APP-containing transgene, e.g., micebearing a 717 mutation of APP described by Games et al., Nature 373:523–527, 1995 and Wadsworth et al. (U.S. Pat. No. 5,811,633, U.S. Pat.No. 5,604,131, U.S. Pat. No. 5,720,936), and mice bearing a Swedishmutation of APP such as described by McConlogue et al. (U.S. Pat. No.5,612,486) and Hsiao et al. (U.S. Pat. No. 5,877,399); Staufenbiel etal., Proc. Natl. Acad. Sci. USA 94, 13287–13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287–13292(1997); Borchelt et al., Neuron 19, 939–945 (1997), all of which areincorporated herein by reference.

Compounds or agents found to be efficacious and safe in such animalmodels will be further tested in standard toxicological assays.Compounds showing appropriate toxicological and pharmacokinetic profileswill be moved into human clinical trials for treatment of Alzheimer'sdisease and related diseases. The same screening approach can be used onother potential agents such as peptidomimetics described above.

In general, in selecting therapeutic compounds based on the foregoingassays, it is useful to determine whether the test compound has anacceptable toxicity profile, e.g., in a variety of in vitro cells andanimal model(s). It may also be useful to search the tested andidentified compound(s) against existing compound databases to determinewhether the compound or analogs thereof have been previously employedfor pharmaceutical purposes, and if so, optimal routes of administrationand dose ranges. Alternatively, routes of administration and dosageranges can be determined empirically, using methods well known in theart (see, e.g., Benet, L. Z., et al. Pharmacokinetics in Goodman &Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition,Hardman, J. G., et al., Eds., McGraw-Hill, New York, 1966) applied tostandard animal models, such as a transgenic PDAPP animal model (e.g.,Games, D., et al. Nature 373: 523–527, 1995; Johnson-Wood, K., et al.,Proc. Natl. Acad. Sci. USA 94: 1550–1555, 1997). To optimize compoundactivity and/or specificity, it may be desirable to construct a libraryof near-neighbor analogs to search for analogs with greater specificityand/or activity. Methods for synthesizing near-neighbor and/or targetedcompound libraries are well-known in the combinatorial library field.

C. Inhibitors and Therapeutics

Part B, above, describes method of screening for compounds havingβ-secretase inhibitory activity. To summarize, guidance is provided forspecific methods of screening for potent and selective inhibitors ofβ-secretase enzyme. Significantly, the practitioner is directed to aspecific peptide substrate/inhibitor sequences, such as P10-P4′staD→V(SEQ ID NO:72), on which drug design can be based and additionalsources, such as biased phage display libraries, that should provideadditional lead compounds.

The practitioner is also provided ample guidance for further refinementof the binding site of the enzyme, for example, by crystallizing thepurified enzyme in accord with the methods provide herein. Noting thesuccess in this area that has been enjoyed in the area of HIV proteaseinhibitor development, it is contemplated that such efforts will lead tofurther optimization of the test compounds described herein. Withoptimized compounds in hand, it is possible to define a compoundpharmacophore, and further search existing pharmacophore databases,e.g., as provided by Tripos, to identify other compounds that may differin 2-D structural formulae with the originally discovered compounds, butwhich share a common pharmacophore structure and activity. Testcompounds are assayed in any of the inhibitor assays described herein,at various stages in development. Therefore, the present inventionincludes β-secretase inhibitory agents discovered by any of the methodsdescribed herein, particularly the inhibitor assays and thecrystallization/optimization protocols. Such inhibitory agents aretherapeutic candidates for treatment of Alzheimer's disease, as well asother amyloidoses characterized by Aβ peptide deposition. Theconsiderations concerning therapeutic index (toxicology),bioavailability and dosage discussed in Part B above are also importantto consider with respect to these therapeutic candidates.

D. Methods of Diagnosis

The present invention also provides methods of diagnosing individualswho carry mutations that provide enhanced β-secretase activity. Forexample, there are forms of familial Alzheimer's disease in which theunderlying genetic disorder has yet to be recognized. Members offamilies possessing this genetic predisposition can be monitored foralterations in the nucleotide sequence that encodes β-secretase and/orpromoter regions thereof, since it is apparent, in view of the teachingsherein, that individuals who overexpress of the enzyme or possesscatalytically more efficient forms of the enzyme would be likely toproduce relatively more Aβ peptide. Support for this supposition isprovided by the observation, reported herein, that the amount ofβ-secretase enzyme is rate limiting for production of Aβ in cells.

More specifically, persons suspected to have a predilection fordeveloping for developing or who already have the disease, as well asmembers of the general population, may be screened by obtaining a sampleof their cells, which may be blood cells or fibroblasts, for example,and testing the samples for the presence of genetic mutations in theβ-secretase gene, in comparison to SEQ ID NO: 1 described herein, forexample. Alternatively or in addition, cells from such individuals canbe tested for β-secretase activity. According to this embodiment, aparticular enzyme preparation might be tested for increased affinityand/or Vmax with respect to a β-secretase substrate such as MBP-C125, asdescribed herein, with comparisons made to the normal range of valuesmeasured in the general population. Individuals whose β-secretaseactivity is increased compared to normal values are susceptible todeveloping Alzheimer's disease or other amyloidogenic diseases involvingdeposition of Aβ peptide.

E. Therapeutic Animal Models

A further utility of the present invention is in creation of certaintransgenic and/or knockout animals that are also useful in the screeningassays described herein. Of particular use is a transgenic animal thatoverexpresses the β-secretase enzyme, such as by adding an additionalcopy of the mouse enzyme or by adding the human enzyme. Such an animalcan be made according to methods well known in the art (e.g., Cordell,U.S. Pat. No. 5,387,742; Wadsworth et al., U.S. Pat. No. 5,811,633, U.S.Pat. No. 5,604,131, U.S. Pat. No. 5,720;936; McConlogue et al., U.S.Pat. No. 5,612,486; Hsiao et al., U.S. Pat. No. 5,877,399; and“Manipulating the Mouse Embryo, A Laboratory Manual,” B. Hogan, F.Costantini and E. Lacy, Cold Spring Harbor Press, 1986)), substitutingthe one or more of the constructs described with respect to β-secretase,herein, for the APP constructs described in the foregoing references,all of which are incorporated by reference.

An overexpressing β-secretase transgenic mouse will make higher levelsof Aβ and sβAPP from APP substrates than a mouse expressing endogenousβ-secretase. This would facilitate analysis of APP processing andinhibition of that processing by candidate therapeutic agents. Theenhanced production of Aβ peptide in mice transgenic for β-secretasewould allow acceleration of AD-like pathology seen in APP transgenicmice. This result can be achieved by either crossing the β-secretaseexpressing mouse onto a mouse displaying AD-like pathology (such as thePDAPP or Hsiao mouse) or by creating a transgenic mouse expressing boththe β-secretase and APP transgene.

Such transgenic animals are used to screen for β-secretase inhibitors,with the advantage that they will test the ability of such inhibitors togain entrance to the brain and to effect inhibition in vivo.

Another animal model contemplated by the present invention is aso-called “knock-out mouse” in which the endogenous enzyme is eitherpermanently (as described in U.S. Pat. Nos. 5,464,764, 5,627,059 and5,631,153, which are incorporated by reference in their entity) orinducibly deleted (as described in U.S. Pat. No. 4,959,317, which isincorporated by reference in its entity), or which is inactivated, asdescribed in further detail below. Such mice serve as controls forβ-secretase activity and/or can be crossed with APP mutant mice, toprovide validation of the pathological sequelae. Such mice can alsoprovide a screen for other drug targets, such as drugs specificallydirected at Aβ deposition events.

β-secretase knockout mice provide a model of the potential effects ofβ-secretase inhibitors in vivo. Comparison of the effects of β-secretasetest inhibitors in vivo to the phenotype of the β-secretase knockout canhelp guide drug development. For example, the phenotype may or may notinclude pathologies seen during drug testing of β-secretase inhibitors.If the knockout does not show pathologies seen in the drug-treated mice,one could infer that the drug is interacting non-specifically withanother target in addition to the β-secretase target. Tissues from theknockout can be used to set up drug binding assays or to carry outexpression cloning to find the targets that are responsible for thesetoxic effects. Such information can be used to design further drugs thatdo not interact with these undesirable targets. The knockout mice willfacilitate analyses of potential toxicities that are inherent toβ-secretase inhibition. Knowledge of potential toxicities will helpguide the design of design drugs or drug-delivery systems to reduce suchtoxicities. Inducible knockout mice are particularly useful indistinguishing toxicity in an adult animal from embryonic effects seenin the standard knockout. If the knockout confers fetal-lethal effects,the inducible knockout will be advantageous.

Methods and technology for developing knock-out mice have matured to thepoint that a number of commercial enterprises generate such mice on acontract basis (e.g., Lexicon Genetics, Woodland Tex.; Cell & MolecularTechnologies, Lavallette, N.J.; Crysalis, DNX Transgenic Sciences,Princeton, N.J.). Methodologies are also available in the art. (SeeGalli-Taliadoros, L. A., et al., J. Immunol. Meth. 181: 1–15, 1995).Briefly, a genomic clone of the enzyme of interest is required. Where,as in the present invention, the exons encoding the regions of theprotein have been defined, it is possible to achieve inactivation of thegene without further knowledge of the regulatory sequences controllingtranscription. Specifically, a mouse strain 129 genomic library can bescreened by hybridization or PCR, using the sequence informationprovided herein, according to methods well known in the art. (Ausubel;Sambrook) The genomic clone so selected is then subjected to restrictionmapping and partial exonic sequencing for confirmation of mousehomologue and to obtain information for knock-out vector construction.Appropriate regions are then sub-cloned into a “knock-out” vectorcarrying a selectable marker, such as a vector carrying a neo^(r)cassette, which renders cells resistant to aminoglycoside antibioticssuch as gentamycin. The construct is further engineered for disruptionof the gene of interest, such as by insertion of a sequence replacementvector, in which a selectable marker is inserted into an exon of thegene, where it serves as a mutagen, disrupting the coordinatedtranscription of the gene. Vectors are then engineered for transfectioninto embryonic stem (ES) cells, and appropriate colonies are isolated.Positive ES cell clones are micro-injected into isolated hostblastocysts to generate chimeric animals, which are then bred andscreened for germline transmission of the mutant allele.

According to a further preferred embodiment, β-secretase knock-out micecan be generated such that the mutation is inducible, such as byinserting in the knock-out mice a lox region flanking the β-secretasegene region. Such mice are then crossed with mice bearing a “Cre” geneunder an inducible promoter, resulting in at least some off-springbearing both the “Cre” and the lox constructs. When expression of “Cre”is induced, it serves to disrupt the gene flanked by the lox constructs.Such a “Cre-lox” mouse is particularly useful, when it is suspected thatthe knock-out mutation may be lethal. In addition, it provides theopportunity for knocking out the gene in selected tissues, such as thebrain. Methods for generating Cre-lox constructs are provided by U.S.Pat. No. 4,959,317, incorporated herein by reference, and are made on acontractual basis by Lexicon Genetics, Woodlands, Tex., among others.

The following examples illustrate, but in no way are intended to limitthe present invention.

EXAMPLE 1 Isolation of Coding Sequences for Human β-secretase

A. PCR Cloning

Poly A+ RNA from IMR human neuroblastoma cells was reverse transcribedusing the Perkin-Elmer kit. Eight degenerate primer pools, each 8 folddegenerate, encoding the N and C terminal portions of the amino acidsequence obtained from the purified protein were designed (shown inTable 4; oligos 3407 through 3422) (SEQ ID NOS:3–21). PCR reactions werecomposed of cDNA from 10 ng of RNA, 1.5 mM MgCl₂, 0.125 μl AmpliTaqGold, 160 μM each dNTP (plus 20 μM additional from the reversetranscriptase reaction), Perkin-Elmer TAQ buffer (from AmpliTaq Goldkit, Perkin-Elmer, Foster City, Calif.), in a 25 μl reaction volume.Each of oligonucleotide primers 3407 through 3414 was used incombination with each of oligos 3415 through 3422 for a total for 64reactions. Reactions were run on the Perkin-Elmer 7700 SequenceDetection machine under the following conditions: 10 min at 95° C., 4cycles of, 45° C. annealing for 15 seconds, 72° C. extension for 45seconds and 95° C. denaturation for 15 seconds followed by 35 cyclesunder the same conditions with the exception that the annealingtemperature was raised to 55° C. (The foregoing conditions are referredto herein as “Reaction 1 conditions.”) PCR products were visualized on4% agarose gel (Northern blots) and a prominent band of the expectedsize (68 bp) was seen in reactions, particularly with the primers3515–3518. The 68 kb band was sequenced and the internal region codedfor the expected amino acid sequence. This gave the exact DNA sequencefor 22 bp of the internal region of this fragment.

Additional sequence was deduced from the efficiency of various primerpools of discrete sequence in generating this PCR product. Primer pools3419 to 3422 (SEQ ID NOS:15–18) gave very poor or no product, whereaspools 3415 to 3418 (SEQ ID NOS:11–14 respectively) gave robust signal.The difference between these pools is a CTC (3415 to 3418) (SEQ IDNOS:11–14) vs TTC (3419 to 3422) (SEQ ID NOS:15–18) in the 3′ most endof the pools. Since CTC primed more efficiently we can conclude that thereverse complement GAG is the correct codon. Since Met coding is uniqueit was concluded that the following codon is ATG. Thus the exact DNAsequence obtained is:

CCC. GGC. CGG. AGG. GGC. AGC. TTT. GTG. GAG. ATG. GT (SEQ ID NO: 49)encoding the amino acid sequence P G R R G S F V E M V (SEQ ID NO: 50).This sequence can be used to design exact oligonucleotides for 3 and 5′RACE PCR on either cDNA or libraries or to design specific hybridizationprobes to be used to screen libraries.

Since the degenerate PCR product was found to be so robust, thisreaction may also be used as a diagnostic for the presence of clonescontaining this sequence. Pools of libraries can be screened using thisPCR product to indicate the presence of a clone in the pool. The poolscan be broken out to identify individual clones. Screening pools ofknown complexity and or size can provide information on the abundance ofthis clone in a library or source and can approximate the size of thefull length clone or message.

For generation of a probe, PCR reactions using oligonucleotides 3458(SEQ ID NO:19) and 3469 (SEQ ID NO:21) or 3458 (SEQ ID NO: 19) and 3468(SEQ ID NO: 20) (Table 4) can be carried out using the 23 RACE product,clone 9C7E.35 (30 ng, clone 9C7E.35 was isolated from origene library,see Example 2), or cDNA generated from brain, using the standard PCRconditions (Perkin-Elmer, rtPCR and AmpliTaq® Gold kits) with thefollowing 25 μl reaction volume 1.5 mM MgCl₂, 0.125 μl of AmpliTaq® Gold(Perkin-Elmer), initial 95° for 10 min to activate the AmpliTaq® Gold,36 cycles of 65° for 15 sec, 72° for 45 sect 95° for 15 sec, followed by3 min at 72°. Product was purified on a Quiagen PCR purification kit andused as a substrate for randompriming to generate a radiolabelled probe(Sambrook, et al., supra; Amersham RediPrime® kit). This probe was usedto isolate full length close pCEK clone 27 shown in FIGS. 12 and 13(A–E) (SEQ ID NO:48).

Derivation of Full Length Clone pCEK Clone 27

A human primary neuronal cell library in the mammalian expression vectorpCEK2 vector was generated using size selected cDNA, and pools of clonesgenerated from different sized inserts. The cDNA library for13β-secretase screening was made with poly(A)⁺ RNA isolated from primaryhuman neuronal cells. The cloning vector was pCEK2 (FIG. 12).

pCEK2

Double-stranded cDNA inserts were synthesized using the cDNA SynthesisKit from Stratagene with some modifications. The inserts were thenfractionated according to their sizes. A total of five fractions wereindividually ligated with double-cut (NotI and XhoI) pCEK2 andsubsequently transformed into the E. Coli strain XL-10 Gold which isdesigned to accept very large plasmids.

The fractions of transformed E. coli were plated on Terrific Broth agarplates containing ampicilin and let grown for 18 hours. Each fractionyielded about 200,000 colonies to give a total of one million colonies.The colonies were then scraped from the plates and plasmids isolatedfrom them in pools of approximately 70,000 clones/pool. 70,000 clonesfrom each pool of the library was screened for the presence of theputative β-secretase gene using the diagnostic PCR reaction (degenerateprimers 3411 (SEQ ID NO:7) and 3417 (SEQ ID NO:13) shown above).

Clones from the 1.5 kb pool were screened using a radiolabeled probegenerated from a 390 b.p. PCR product generated from clone 9C7E.35. Forgeneration of a probe, PCR product was generated using 3458 (SEQ IDNO:19) and 3468 (SEQ ID NO:20) as primers and clone 9C7E.35 (30 ng) assubstrate.

PCR product was used as a substrate for random priming to generate aradiolabeled probe. 180,000 clones from the 1.5 kb pool (70,000 originalclones in this pool), were screened by hybridization with the PCR probeand 9 positive clones identified. Four of these clones were isolated andby restriction mapping these appear to encode two independent clones of4 to 5 kb (clone 27) and 6 to 7 kb (clone 53) length. Sequencing ofclone 27 verified that it contains a coding region of 1.5 kb. FIG. 13(A–E) shows the sequence of pCEK clone 27 (clone 27) (SEQ ID NO:48).

TABLE 4 SEQ ID Pool Nucleotide Sequence NO. No. (Degeneratesubstitutions are shown in parentheses) 3 3407G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAG.GAG.CC 4 3408G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAA.GAG.CC 5 3409G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAA.GAA.CC 6 3410G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAG.GAA.CC 7 3411AGA.GAC.GA(GA).GA(GA).CC(CG).GAG.GAG.CC 8 3412AGA.GAC.GA(GA).GA(GA).CC(CG).GAA.GAG.CC 9 3413AGA.GAC.GA(GA).GA(GA).CC(CG).GAA.GAA.CC 10 3414AGA.GAC.GA(GA).GA(GA).CC(CG)GAG.GAA.CC 11 3415CG.TCA.CAG.(GA)TT.(GA)TC.AAC.CAT.CTC 12 3416CG.TCA.CAG.(GA)TT.(GA)TC.TAC.CAT.CTC 13 3417CG.TCA.CAG.(GA)TT.(GA)TC.CAC.CAT.CTC 14 3418CG.TCA.CAG.(GA)TT.(GA)TC.GAC.CAT.CTC 15 3419CG.TCA.CAG.(GA)TT.(GA)TC.AAC.CAT.TTC 16 3420CG.TCA.CAG.(GA)TT.(GA)TC.TAC.CAT.TTC 17 3421CG.TCA.CAG.(GA)TT.(GA)TC.CAC.CAT.TTC 18 3422CG.TCA.CAG.(GA)TT.(GA)TC.GAC.CAT.TTC 19 3458 GAG GGG CAG CTT TGT GGA GA20 3468 CAG.CAT.AGG.CCA.GCC.CCA.GGA.TGC.CT 21 3469GTG.ATG.GCA.GCA.ATG.TTG.GCA.CGC

EXAMPLE 2 Screening of Human Fetal Brain cDNA Library

The Origene human fetal brain Rapid-Screen™ cDNA Library Panel isprovided as a 96-well format array consisting of 5000 clones (plasmidDNA) per well from a human fetal brain library. Subplates are availablefor each well consisting of 96 wells of 50 clones each in E. coli. Thisis an oligo-dT primed library, size-selected and unidirectionallyinserted into the vector pCMV-XL3.

94 wells from the master plate were screened using PCR. The Reaction 1Conditions described in Example 1, above, were followed, using onlyprimers 3407 (SEQ ID NO:3) and 3416 (SEQ ID NO:12) with 30ng of plasmidDNA from each well. Two pools showed the positive 70 bp band. The sameprimers and conditions were used to screen 1 μl E. coli from each wellof one of the subplates. E. coli from the single positive well was thenplated onto LB/amp plates and single colonies screened using the samePCR conditions. The positive clone, about 1 Kb in size, was labeled9C7E.35. It contained the original peptide sequence as well as 5′sequence that included a methionine. The 3′ sequence did not contain astop codon, suggesting that this was not a full-length clone, consistentwith Northern blot data.

EXAMPLE 3 PCR Cloning Methods

3′RACE was used in experiments carried out in support of the presentinvention to elucidate the polynucleotide encoding human β-secretase.Methods and conditions appropriate for replicating the experimentsdescribed herein and/or determining polynucleotide sequences encodingadditional members of the novel family of aspartyl proteases describedherein may be found, for example, in White, B. A., ed., PCR CloningProtocols; Humana Press, Totowa, N.J., 1997, or Ausubel, supra, both ofwhich are incorporated herein by reference.

RT-PCR

For reverse transcription polymerase chain reaction (RT-PCR), twopartially degenerate primer sets used for RT-PCR amplification of a cDNAfragment encoding this peptide. Primer set 1 consisted of DNA's#3427–3434 (SEQ ID NOS:22–29 respectively), the sequences of which areshown in Table 5, below. Matrix RT-PCR using combinations of primersfrom this set with cDNA reverse transcribed from primary human neuronalcultures as template yielded the predicted 54 bp cDNA product withprimers #3428+3433 (SEQ ID NOS:23–28 respectively. All RT-PCR reactionsemployed 10–50 ng input poly-A+ RNA equivalents per reaction, and werecarried out for 35 cycles employing step cycle conditions with a 95° C.denaturation for 1 minute, 50° C. annealing for 30 sec, and a 72° C.extension for 30 sec.

The degeneracy of primers #3428+3433 (SEQ ID NOS:23–28) was furtherbroken down, resulting in primer set 2, comprising DNAs #3448–3455 (SEQID NOS:30–37) (Table 5). Matrix RT-PCR was repeated using primer set 2,and cDNA reverse transcribed from poly-A+ RNA from IMR-32 humanneuroblastoma cells (American Type Culture Collection, Manassas, Va.),as well as primary human neuronal cultures, as template foramplification. Primers #3450 (SEQ ID NO:32) and 3454 (SEQ ID NO:36) fromset 2 most efficiently amplified a cDNA fragment of the predicted size(72 bp), although primers 3450+3453 (SEQ ID NOS:32 and 35), and3450+3455 (SEQ ID NOS:32 and 37) also amplified the same product, albeitat lower efficiency. A 72 bp PCR product was obtained by amplificationof cDNA from IMR-32 cells and primary human neuronal cultures withprimers 3450 (SEQ ID NO:32) and 3454 (SEQ ID NO:36).

Internal primers matching the upper (coding) strand for 3′ RapidAmplification of 5′ Ends (RACE) PCR, and lower (non-coding) strand for5′ RACE PCR were designed and made according to methods known in the art(e.g., Frohman, M. A., M. K. Dush and G. R. Martin (1988). “Rapidproduction of full-length cDNAs from rare transcripts: amplificationusing a single gene specific oligo-nucleotide primer.” Proc. Natl. Acad.Sci. U.S.A. 85(23): 8998–9002.) The DNA primers used for this experiment(#3459 & #3460) (SEQ ID NOS:38 and 39) are illustrated schematically inFIG. 4, and the exact sequence of these primers is presented in Table 3.These primers can be utilized in standard RACE-PCR methodology employingcommercially available templates (e.g. Marathon Ready cDNA®, ClontechLabs), or custom tailored cDNA templates prepared from RNAs of interestas described by Frohman et al. (ibid.).

In experiments carried out in support of the present invention, avariation of RACE was employed to exploit an IMR-32 cDNA library clonedin the retrovirus expression vector pLPCXlox, a derivative of pLNCX. Asthe vector junctions provide unique anchor sequences abutting the cDNAinserts in this library, they serve the purpose of 5′ and 3′ anchorprimers in RACE methodology. The sequences of the specific 5′ and 3′anchor primers we employed to amplify β-secretase cDNA clones from thelibrary, primers #3475 (SEQ ID NO:40) and #3476 (SEQ ID NO:41), arederived from the DNA sequence of the vector provided by Clontech Labs,Inc., and are shown in Table 3.

Primers #3459 (SEQ ID NO:38) and #3476 (SEQ ID NO:41) were used for 3′RACE amplification of downstream sequences from our IMR-32 cDNA libraryin the vector pLPCXlox. The library had previously been sub-divided into100 pools of 5,000 clones per pool, and plasmid DNA was isolated fromeach pool. A survey of the 100 pools with the primers identified asdiagnostic for presence of the β-secretase clone, according to methodsdescribed in Example 1, above, provided individual pools from thelibrary for RACE-PCR. 100 ng template plasmid from pool 23 was used forPCR amplification with primers 3459+3476 (SEQ ID NOS:38 and 41respectively). Amplification was carried out for 40 cycles usingampli-Taq Gold®, under the following conditions: denaturation at 95° C.for 1 min, annealing at 65° C. for 45 sec., and extension at 72° C. for2 min. Reaction products were fractionated by agarose gelchromatography, according to methods known in the art (Ausubel;Sambrook).

An approximately 1.8 Kb PCR fragment was revealed by agarose gelfractionation of the reaction products. The PCR product was purifiedfrom the gel and subjected to DNA sequence analysis using primer#3459(SEQ ID NO:38). The resulting sequence, designated 23A, and thepredicted amino acid sequence deduced from the DNA sequence are shown inFIG. 5. Six of the first seven deduced amino-acids from one of thereading frames of 23A were an exact match with the last 7 amino-acids ofthe N-terminal sequence determined from the purified protein, purifiedand sequenced in further experiments carried out in support of thepresent invention, from natural sources.

TABLE 5 SEQ ID NO. DNA # NUCLEOTIDE SEQUENCE COMMENTS 22 3427 GAY GARGAG CCN GAG GA 23 3428 GAY GAR GAG CCN GAa GA 24 3429 GAY GAR GAa CCNGAg GA 25 3430 GAY GAR GAa CCN GAa GA 26 3431 RTT RTC NAC CAT TTC 273432 RTT RTC NAC CAT cTG 28 3433 TCN ACC ATY TCN ACA AA 29 3434 TCN ACCATY TCN ACG AA 30 3448 ata ttc tag a GAY GAR GAg CCa GAa GA 5′ primer,break down of 3428 w/ 5′ Xbal tail, 1 of 4 31 3449 ata ttc tag a GAY GARGAg CCg GAa GA 5′ primer, break down of 3428 w/ 5′ Xbal tail, 2 of 4 323450 ata ttc tag a GAY GAR GAg CCc GAa GA 5′ primer, break down of 3428w/ 5′ Xbal tail, 3 of 4 33 3451 ata ttc tag a GAY GAR GAg CCt GAa GA 5′primer, break down of 3428 w/ 5′ Xbal tail, 4 of 4 34 3452 aca cga att cTT RTC NAC CAT YTC aAG AAA breakdown of 3433, 1 of 4; tm = 50 35 3453aca cga att c TT RTC NAC CAT YTC gAC AAA breakdown of 3433 w/ 5′ Eco RItail, 2 of 4; tm = 50 36 3454 aca cga att c TT RTC NAC CAT YTC cAC AAAbreakdown of 3433 w/ 5′ Eco RI tail, 3 of 4; tm = 50 37 3455 acacga att c TT RTC NAC CAT YTC tAC AAA breakdown of 3433 w/ 5′ Eco RItail, 4 of 4; tm = 50 38 3459 aa gaG CCC GGC CGG AGG GGC A 5′ upperstrand primer for 3′ race encodes eEPGRRG 39 3460 aaa GCT GCC CCT CCGGCC GGG 3′ lower strand primer for 5′ RACE 40 3475 AGC TCG TTT AGT GAACCG TCA GAT CG pLNCX 5′ primer 41 3476 ACC TAC AGG TGG GGT CTT TCA TTCCC pLNCX, 3′ primer

EXAMPLE 4 β-secretase Inhibitor Assays

Assays for measuring β-secretase activity are well known in the art.Particularly useful assays, summarized below, are detailed in allowedU.S. Pat. No. 5,744,346, incorporated herein by reference.

A. Preparation of MBP-C125sw

1. Preparation of Cells

Two 250 ml cell culture flasks containing 50 ml LBamp100 per flask wereseeded with one colony per flask of E. coli pMAL-C125SW cl. 2 (E. coliexpressing MBP-C125sw fusion protein). Cells were allowed to growovernight at 37° C. Aliqouts (25 ml) were seeded in 500 ml per flask ofLBamp100 in 2 liter flasks, which were then allowed to grow at 30°.Optical densities were measured at 600 nm (OD₆₀₀) vs LB broth; 1.5 ml100 mM IPTG was added when the OD was ^(˜)0.5. At this point, apre-incubation aliqout was removed for SDS-PAGE (“−1”). Of this aliqout,0.5 ml was centrifuged for 1 min in a Beckman microfuge, and theresulting pellet was dissolved in 0.5 ml 1×LSB. The cells wereincubated/induced for 5–6 hours at 30 C, after which a post-incubationaliquot (“+I”) was removed. Cells were then centrifuged at 9,000 rpm ina KA9.1 rotor for 10 min at 4° C. Pellets were retained and stored at−20 C.

2. Extraction of Bacterial Cell Pellets

Frozen cell pellets were resuspended in 50 ml 0.2 M NaCl, 50nM Tris, pH7.5, then sonicated in rosette vessal for 5×20 sec bursts, with 1 minrests between bursts. The extract was centrifuged at 16,500 rpm in aKA18.5 rotor 30 min (39,000×g). Using pipette as a pestle, the sonicatedpellet was suspended in 50 ml urea extraction buffer (7.6 M urea, 50 mMTris pH 7.5, 1 mM EDTA, 0.5% TX-100). The total volume was about 25 mlper flask. The suspension was then sonicated 6×20 sec, with 1 min restsbetween bursts. The suspension was then centrifuged again at 16,500 rpm30 m in the KA18.5 rotor. The resulting supernatant was added to 1.5 Lof buffer consisting of 0.2 M NaCl 50 mM Tris buffer, pH 7.5, with 1%Triton X-100 (0.2M NaCl-Tris-1% Tx), and was stirred gently at 4 degreesC. for 1 hour, followed by centrifugation at 9,000 rpm in KA9.1 for 30min at 4° C. The supernatant was loaded onto a column of washed amylose(100 ml of 50% slurry; New England BioLabs). The column was washed with0.2 M NaCl-Tris-1% TX to baseline (+10 column volumes), then with 2column volumes 0.2M NaCl-Tris-1% reduced Triton X-100. The protein wasthen eluted with 10 mM maltose in the same buffer. An equal volume of 6M guanidine HCl/0.5% TX-100 was added to each fraction. Peak fractionswere pooled and diluted to a final concentration of about 2 mg/ml. Thefractions were stored at −40 degrees C., before dilution (20-fold, to0.1 mg/ml in 0.15% Triton X-100). Diluted aliquots were also stored at−40 C.

B. Antibody-based Assays

The assays described in this section are based on the ability of certainantibodies, hereinafter “cleavage-site antibodies,” to distinguishcleavage of APP by β-secretase, based on the unique cleavage site andconsequent exposure of a specific C-terminus formed by the cleavage. Therecognized sequence is a sequence of usually about 3–5 residues isimmediately amino terminal of the p amyloid peptide (PAP) produced byβ-secretase cleavage of β-APP, such as Val-Lys-Met in wild-type orVal-Asn-Leu- in the Swedish double mutation variant form of APP.Recombinantly-expressed proteins, described below, were used assubstrates for β-secretase.

MBP-C125 Assay: MBP-C125 substrates were expressed in E. coli as afusion protein of the last 125 amino acids of APP fused to thecarboxy-terminal end of maltose-binding protein (MBP), usingcommercially available vectors from New England Biolabs. The β-cleavagesite was thus 26 amino acids downstream of the start of the C-125region. This latter site is recognized by monoclonal antibody SW192.

Recombinant proteins were generated with both the wild-type APP sequence(MBP-C125 wt) at the cleavage site ( . . . Val-Lys-Met-Asp-Ala . . . )(SEQ ID NO: 54) or the “Swedish” double mutation (MBP-C125 sw) ( . . .Val-Asn-Leu-Asp-Ala . . . ) (SEQ ID NO: 51). As shown schematically inFIG. 19A, cleavage of the intact MBP-fusion protein results in thegeneration of a truncated amino-terminal fragment, with the new SW-192Ab-positive epitope uncovered at the carboxy terminus. Thisamino-terminal fragment can be recognized on Western blots with the sameAb, or, quantitatively, using an anti-MBP capture-biotinylated SW-192reporter sandwich format, as shown in FIG. 19A. Anti-MBP polyclonalantibodies were raised in rabbits (Josman Labs, Berkeley) byimmunization with purified recombinantly expressed MBP (New EnglandBiolabs). Antisera were affinity purified on a column of immobilizedMBP. MBP-C125 SW and WT substrates were expressed in E. coli, thenpurified as described above.

Microtiter 96-well plates were coated with purified anti-MBP antibody(at a concentration of 5–10 μg/ml), followed by blocking with 2.5g/liter human serum albumin in 1 g/liter sodium phosphate monobasic,10.8 g/liter sodium phosphate dibasic, 25 g/liter sucrose, 0.5 g/litersodium azide, pH 7.4. Appropriately diluted β-secretase enzyme (5 μl)was mixed with 2.5 μl of 2.2 μM MBP-C125sw substrate stock, in a 50 μlreaction mixture with a final buffer concentration of 20 mM acetatebuffer, pH 4.8, 0.06% Triton X-100, in individual wells of a 96-wellmicrotiter plate, and incubated for 1 hour at 37 degrees C. Samples werethen diluted 5-fold with Specimen Diluent (0.2 g/l sodium phosphatemonobasic, 2.15 g/l sodium phosphate dibasic, 0.5 g/l sodium azide, 8.5g/l sodium chloride, 0.05% Triton X-405, 6 g/l BSA), further diluted5–10 fold into Specimen Diluent on anti-MBP coated plates, and incubatedfor 2 hours at room temperature. Following incubations with samples orantibodies, plates were washed at least four times in TTBS (0.15 M NaCl,50 mM Tris, ph&0.5, 0.05% Tween-20). Biotinylated SW192 antibodies wereused as the reporter. SW192 polyclonal antibodies were biotinylatedusing NHS-biotin (Pierce), following the manufacturer's instruction.Usually, the biotinylated antibodies were used at about 240 ng/ml, theexact concentration varying with the lot of antibodies used. Followingincubation of the plates with the reporter, the ELISA was developedusing streptavidin-labeled alkaline phosphatase (Boeringer-Mannheim) and4-methyl-umbelliferyl phosphate as fluorescent substrate. Plates wereread in a Cytofluor 2350 Fluorescent Measurement System. Recombinantlygenerated MBP-26SW (product analog) was used as a standard to generate astandard curve, which allowed the conversion of fluorescent units intoamount of product generated.

This assay protocol was used to screen for inhibitor structures, using“libraries” of compounds assembled onto 96-well microtiter plates.Compounds were added, in a final concentration of 20 μg/ml in 2% DMSO,in the assay format described above, and the extent of product generatedcompared with control (2% DMSO only) α-secretase incubations, tocalculate “% inhibition.” “Hits” were defined as compounds which resultin >35% inhibition of enzyme activity at test concentration. This assaycan also be used to provide IC₅₀ values for inhibitors, by varying theconcentration of test compund over a range to calculate from adose-response curve the concentration required to inhibit the activityof the enzyme by 50%.

Generally, inhibition is considered significant as compared to controlactivity in this assay if it results in activity that is at least 1standard deviation, and preferably 2 standard deviations lower than amean activity value determined over a range of samples. In addition, areduction of activity that is greater than about 25%, and preferablygreater than about 35% of control activity may also be consideredsignificant.

Using the foregoing assay system, 24 “hits” were identified (>30%inhibition at 50 μM concentration) from the first 6336 compounds tested(0.4% hit rate). Of these 12 compounds had IC₅₀s less than 50 μM,including re-screening in the P26-P4′sw assay, below.

P26-P4′sw assay. The P26-P4′sw substrate is a biotin-linked peptide ofthe sequence (biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNLDAEF (SEQ ID NO: 63).The P26-P1 standard has the sequence(biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNL (SEQ ID NO: 64), where theN-terminal “CGG” serves as a linker between biotin and the substrate inboth cases. Peptides were prepared by Anaspec, Inc. (San Jose, Calif.)using solid phase synthesis with boc-amino acids. Biotin was coupled tothe terminal cysteine sulfhydryl by Anaspec, Inc. after synthesis of thepeptide, using EZ-link Iodoacetyl-LC-Biotin (Pierce). Peptides arestored as 0.8–1.0 mM stocks in 5 mM Tris, with the pH adjucted to aroundneutral (pH 6.5–7.5) with sodium hydroxide.

For the enzyme assay, the substrate concentration can vary from 0–200μM. Specifically for testing compounds for inhibitory activity,substrate concentration is 1.0 μM. Compounds to be tested were added inDMSO, with a final DMSO concentration of 5%; in such experiments, thecontrols also receive 5% DMSO. Concentration of enzyme was varied, togive product concentrations within the linear range of the ELISA assay(125–2000 μM, after dilution). These components were incubated in 20 mMsodium acetate, pH 4.5, 0.06% Triton X-100, at 37° C. for 1 to 3 hours.Samples were diluted 5-fold in specimen diluent (145.4 mM sodiumchloride, 9.51 mM sodium phosphate, 7.7 mM sodium azide, 0.05% TritonX-405, 6 μm/liter bovine serum albumin, pH 7.4) to quench the reaction,then diluted further for the ELISA as needed. For the ELISA, Costar HighBinding 96-well assay plates (Corning, Inc., Corning, N.Y.) were coatedwith SW 192 monoclonal antibody from clone 16A7, or a clone of similaraffinity. Biotin-P26-P4′ standards were diluted in specimen diluent to afinal concentration of 0 to 2 nM. Diluted samples and standards (100 μl)are incubated on the SW192 plates at 4° C. for 24 hours. The plates arewashed 4 times in TTBS buffer (150 mM sodium chloride, 25 mM Tris, 0.05%Tween 20, pH 7.5), then incubated with 0.1 ml/well ofstreptavidin—alkaline phosphatase (Roche Molecular Biochemicals,Indianapolis, Ind.) diluted 1:3000 in specimen diluent. After incubatingfor one hour at room temperature, the plate was washed 4 times in TTBS,as described in the previous section, and incubated with fluorescentsubstrate solution A (31.2 μm/liter 2-amino-2-methyl-1-propanol, 30mg/liter, adjusted to pH 9.5 with HCl). Fluorescent values were readafter 30 minutes.

C. Assays Using Synthetic Oligopeptide Substrates

This assay format is particularly useful for measuring activity ofpartially purified α-secretase preparations. Synthetic oligopeptides areprepared which incorporate the known cleavage site of β-secretase, andoptional detectable tags, such as fluorescent or chromogenic moieties.Examples of such peptides, as well as their production and detectionmethods are described in allowed U.S. Pat. No. 5,942,400, hereinincorporated by reference. Cleavage products can be detected using highperformance liquid chromatography, or fluorescent or chromogenicdetection methods appropriate to the peptide to be detected, accordingto methods well known in the art. By way of example, one such peptidehas the sequence SEVNL DAEF (SEQ ID NO: 52), and the cleavage site isbetween residues 5 and 6. Another preferred substrate has the sequenceADRGLTTRPGSGLTNIKTEEISEVNLDAEF (SEQ ID NO: 53), and the cleavage site isbetween residues 26 and 27.

D. β-secretase Assays of Crude Cell or Tissue Extracts

Cells or tissues were extracted in extraction buffer (20 mM HEPES, pH7.5°, 2 mM EDTA, 0.2% Triton X-100, 1 mM PMSF, 20 μg/ml pepstatin, 10μg/ml E-64). The volume of extraction buffer will vary between samples,but should be at least 200 μl per 10⁶ cells. Cells can be suspended bytrituration with a micropipette, while tissue may requirehomogenization. The suspended samples were incubated for 30 minutes onice. If necessary to allow pipetting, unsolubilized material was removedby centrifugation at 4 degrees C., 16,000×g (14,000 rpm in a Beckmanmicrofuge) for 30 minutes. The supernate was assayed by dilution intothe final assay solution. The dilution of extract will vary, but shouldbe sufficient so that the protein concentration in the assay is notgreater than 60 μg/ml. The assay reaction also contained 20 mM sodiumacetate, pH 4.8, and 0.06% Triton X-100 (including Triton contributed bythe extract and substrate), and 220–110 nM MBP-C125 (a 1:10 or 1:20dilution of the 0.1 mg/ml stock described in the protocol for substratepreparation). Reactions were incubated for 1–3 hours at, 37 degrees C.before quenching with at least 5-fold dilution in specimen diluent andassaying using the standard protocol.

EXAMPLE 5 Purification of β-secretase

A. Purification of Naturally Occurring β-secretase

Human 293 cells were obtained and processed as described in U.S. Pat.No. 5,744,346, incorporated herein by reference. (293 cells areavailable from the American Type Culture Collection, Manassas, Va.).Frozen tissue (293 cell paste or human brain) was cut into pieces andcombined with five volumes of homogenization buffer (20 mM Hepes, pH7.5, 0.25 M sucrose, 2 mM EDTA). The suspension was homogenized using ablender and centrifuged at 16,000×g for 30 min at 4° C. The supernatantswere discarded and the pellets were suspended in extraction buffer (20mM MES, pH 6.0, 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA, 5 μg/mlleupeptin, 5 μg/ml E64, 1 μg/ml pepstatin, 0.2 mM PMSF) at the originalvolume. After vortex-mixing, the extraction was completed by agitatingthe tubes at 4° C. for a period of one hour. The mixtures werecentrifuged as above at 16,000×g, and the supernatants were pooled. ThepH of the extract was adjusted to 7.5 by adding ˜1% (v/v) of 1 M Trisbase (not neutralized).

The neutralized extract was loaded onto a wheat germ agglutinin-agarose(WGA-agarose) column pre-equilibrated with 10 column volumes of 20 mMTris, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA, at 4° C. Onemilliliter of the agarose resin was used for every 1 g of originaltissue used. The WGA-column was washed with 1 column volume of theequilibration buffer, then 10 volumes of 20 mM Tris, pH 7.5, 100 mMNaCl, 2 mM NaCl, 2 mM EDTA, 0.2% Triton X-100 and then eluted asfollows. Three-quarter column volumes of 10% chitin hydrolysate in 20 mMTris, pH 7.5, 0.5%, 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA werepassed through the column after which the flow was stopped for fifteenminutes. An additional five column volumes of 10% chitin hydrolysatesolution were then used to elute the column. All of the above eluateswere combined (pooled WGA-eluate).

The pooled WGA-eluate was diluted 1:4 with 20 mM NaOAc, pH 5.0, 0.5%Triton X-100, 2 mM EDTA. The pH of the diluted solution was adjusted to5.0 by adding a few drops of glacial acetic acid while monitoring thepH. This “SP load” was passed through a 5-ml Pharmacia HiTrap SP-columnequilibrated with 20 mM NaOAc, pH 5.0, 0.5% Triton X-100, 2 mM EDTA, at4 ml/min at 4C.

The foregoing methods provided peak activity having a specific activityof greater than 253 nM product/ml/h/μg protein in the MBP-C125-SW assay,where specific activity is determined as described below, with about1500-fold purification of the protein. Specific activity of the purifiedβ-secretase was measured as follows. MBP C125-SW substrate was combinedat approximately 220 nM in 20 mM sodium acetate, pH 4.8, with 0.06%Triton X-100. The amount of product generated was measured by theβ-secretase assay, also described below. Specific activity was thencalculated as:

${{Specific}{\mspace{11mu}\;}{Activity}} = \frac{\left( {{Product}.\mspace{11mu}{conc}.\mspace{11mu}{nM}} \right)\left( {{Dilution}\mspace{20mu}{factor}} \right)}{\left( {{Enzyme}{\;\;}{{sol}.\;{vol}}} \right)\left( {{{Incub}.{\;\;}{time}}\mspace{14mu} h} \right)\left( {{Enzyme}\mspace{14mu}{{conc}.\mspace{11mu}\text{mg/vol}}} \right)}$

The Specific Activity is thus expressed as pmoles of product producedper μg of β-secretase per hour. Further purification of human brainenzyme was achieved by loading the SP flow through fraction on to theP10-P4′sta D→V affinity column, according to the general methodsdescribed below. Results of this purification step are summarized inTable 1, above.

B. Purification of β-secretase from Recombinant Cells

Recombinant cells produced by the methods described herein generallywere made to over-express the enzyme; that is, they produceddramatically more enzyme per cell than is found to be endogenouslyproduced by the cells or by most tissues. It was found that some of thesteps described above could be omitted from the preparation of purifiedenzyme under these circumstances, with the result that even higherlevels of purification were achieved.

CosA2 or 293 T cells transfected with β-secretase gene construct (seeExample 6) were pelleted, frozen and stored at −80 degrees until use.The cell pellet was resuspended by homogenizing for 30 seconds using ahandheld homogenizer (0.5 ml/pellet of approximately 10⁶ cells inextraction buffer consisting of 20 mM TRIS buffer, pH 7.5, 2 mM EDTA,0.2% Triton X-100, plus protease inhibitors: 5 μg/ml E-64, 10 μg/mlpepstatin, 1 mM PMSF), centrifuged as maximum speed in a microfuge (40minutes at 4 degrees C.). Pellets were suspended in original volume ofextraction buffer, then stirred at 1 hour at 4 degrees C. with rotation,and centrifuted again in a microfuge at maximum speed for 40 minutes.The resulting supernatant was saved as the “extract.” The extract wasthen diluted with 20 mm sodium acetate, pH 5.0, 2 mM EDTA and 0.2%Triton X-100 (SP buffer A), and 5 M NaCl was added to a finalconcentration of 60 mM NaCl. The pH of the solution was then adjusted topH 5.0 with glacial acetic acid diluted 1:10 in water. Aliquots weresaved (“SP load”). The SP load was passed through a 1 ml SP HiTrapcolumn which was pre-washed with 5 ml SP buffer A, 5 ml SP buffer B (SPbuffer A with 1 M NaCl) and 10 ml SP buffer A. An additional 2 ml of 5%SP buffer B was passed through the column to dissplace any remainingsample from the column. The pH of the SP flow-through was adjusted to pH4.5 with 10× diluted acetic acid. This flow-through was then applied toa P10-P4′staD→V-Sepharose Affinity column, as described below. Thecolumn (250 μl bed size) was pre-equilibrated with at least 20 columnvolumes of equilibration buffer (25 mM NaCl, 0.2% Triton X-100, 0.1 mMEDTA, 25 mM sodium acetate, pH 4.5), then loaded with the dilutedsupernatant. After loading, subsequent steps were carried out at roomtemperature. The column was washed with washing buffer (125 mM NaCl,0.2% Triton X-100, 25 mM sodium acetate, pH 4.5) before addition of 0.6column bed volumes of borate elution buffer (200 mM NaCl, 0.2% reducedTriton X-100, 40 mM sodium borate, pH 9.5). The column was then capped,and an additional 0.2 ml elution buffer was added. The column wasallowed to stand for 30 minutes. Two bed volumes elution buffer wereadded, and column fractions (250 μl) were collected The protein peakeluted in two fractions. 0.5 ml of 10 mg/ml peptstatin was added permilliliter of collected fractions.

Cell extracts made from cells transfected with full length clone 27(encoding SEQ ID NO: 2; 1–501), 419stop (SEQ ID NO:57) and 452stop (SEQID NO: 59) were detected by Western blot analysis using antibody 264A(polyclonal antibody directed to amino acids 46–67 of β-secretase withreference to SEQ ID NO: 2).

EXAMPLE 6 Preparation of Heterologous Cells Expressing Recombinantβ-secretase

Two separate clones (pCEKclone27 and pCEKclone53) were transfected into293T or COS(A2) cells using Fugene and Effectene methods known in theart. 293T cells were obtained from Edge Biosystems (Gaithersburg, Md.).They are KEK293 cells transfected with SV40 large antigen. COSA2 are asubclone of COS1 cells; subcloned in soft agar.

FuGENE Method: 293T cells were seeded at 2×10⁵ cells per well of a 6well culture plate. Following overnight growth, cells were atapproximately 40–50% confluency. Media was changed a few hours beforetransfection (2 ml/well). For each sample, 3 μl of FuGENE 6 TransfectionReagent (Roche Molecular Biochemicals, Indianapolis, Ind.) was dilutedinto 0.1 ml of serum-free culture medium (DME with 10 mM Hepes) andincubated at room temperature for 5 min. One microgram of DNA for eachsample (0.5–2 mg/ml) was added to a separate tube. The diluted FuGENEreagent was added drop-wise to the concentrated DNA. After gentletapping to mix, this mixture was incubated at room temperature for 15minutes. The mixture was added dropwise onto the cells and swirledgently to mix. The cells were then incubated at 37 degrees C., in anatmosphere of 7.5% CO₂. The conditioned media and cells were harvestedafter 48 hours. Conditioned media was collected, centrifuged andisolated from the pellet. Protease inhibitors (5 μg/ml E64, 2 μg/mlpeptstatin, 0.2 mM PMSF) were added prior to freezing. The cellmonolayer was rinsed once with PBS, tehn 0.5 ml of lysis buffer (1 mMHIPIS, pH 7.5, 1 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 10 μg/ml E64)was added. The lysate was frozen and thawed, vortex mixed, thencentrifuged, and the supernatant was frozen until assayed.

Effective Method. DNA (0.6 μg) was added with “EFFECTENE” reagent(Qiagen, Valencia, Calif.) into a 6-well culture plate using a standardtransfection protocol according to manufacturer's instructions. Cellswere harvested 3 days after transfection and the cell pellets were snapfrozen. Whole cell lysates were prepared and various amounts of lysatewere tested for β-secretase activity using the MBP-C125sw substrate.FIG. 14B shows the results of these experiments, in which picomoles ofproduct formed is plotted against micrograms of COS cell lysate added tothe reaction. The legend to the figure describes the enzyme source,where activity from cells transfected with DNA from pCEKclone27 andPCEKclone53 (clones 27 and 53) using Effective are shown as closeddiamonds and solid squares, respectively, activity from cellstransfected with DNA from clone 27 prepared with FuGENE are shown asopen triangles, and mock transfected and control plots show no activity(closed triangles and “X” markers). Values greater than 700 μM productare out of the linear range of the assay.

EXAMPLE 7 Preparation of P10-P4′sta(D→V) Sepharose Affinity Matrix A.Preparation of P10-P4′sta(D→V) Inhibitor Peptide

P10-P4′sta(D→V) has the sequence NH₂-KTEEISEVN[sta]VAEF-COOH (SEQ ID NO:72), where “sta” represents a statine moiety. The synthetic peptide wassynthesized in a peptide synthesizer using boc-protected amino acids forchain assembly. All chemicals, reagents, and boc amino acids werepurchased from Applied Biosystems (ABJ; Foster City, Calif.) with theexception of dichloromethane and N,N-dimethylformamide which were fromBurdick and Jackson. The starting resin, boc-Phe-OCH2-Pam resin was alsopurchased from ABI. All amino acids were coupled following preactivationto the corresponding HOBT ester using 1.0 equivalent of1-hydroxybenzotriazole (HOBT), and 1.0 equivalent ofN,N-dicyclohexylcarbodiimide (DCC) in dimethylformamide. The bocprotecting group on the amino acid α-amine was removed with 50%trifluoroacetic acid in dichloromethane after each coupling step andprior to Hydrogen Fluoride cleavage.

Amino acid side chain protection was as follows: Glu(Bzl), Lys(Cl-CBZ),Ser(OBzl), Thr(OBzl). All other amino acids were used with no furtherside chain protection including boc-Statine.

[(Bzl) benzyl, (CBZ) carbobenzoxy, (C1-CBZ) chlorocarbobenzoxy, (OBzl)O-benzyl]

The side chain protected peptide resin was deprotected and cleaved fromthe resin by reacting with anhydrous hydrogen fluoride (HF) at 0° C. forone hour. This generates the fully deprotecled crude peptide as aC-terminal carboxylic acid.

Following HF treatment, the peptide was extracted from the resin inacetic acid and lyophilized. The crude peptide was then purified usingpreparative reverse phase HPLC on a Vydac C4, 330 Å, 10 μm column 2.2 cmI.D.×25 cm in length. The solvent system used with this column was 0.1%TFA/H2O ([A] buffer) and 0.1% TFA/CH3CN ([B] buffer) as the mobilephase. Typically the peptide was loaded onto the column in 2% [B] at8–10 mL/min. and eluted using a linear gradient of 2% [B] to 60% [B] in174 minutes.

The purified peptide was subjected to mass spectrometry, and analyticalreverse phase HPLC to confirm its composition and purity.

B. Incorporation into Affinity Matrix

All manipulations were carried out at room temperature. 12.5 ml of 80%slurry of NHS-Sepharose (i.e. 10 ml packed volume; Pharmacia,Piscataway, N.J.) was poured into a Bio-Rad EconoColumn (BioRad,Richmond, Calif.) and washed with 165 ml of ice-cold 1.0 mM HCl. Whenthe bed was fully drained, the bottom of the column was closed off, and5.0 ml of 7.0 mg/ml P10-P4′sta(D→V) peptide (SEQ ID NO:72) (dissolved in0.1 M HEPES, pH 8.0) was added. The column was capped and incubated withrotation for 24 hours. After incubation, the column was allowed todrain, then washed with 8 ml of 1.0 M ethanolamine, pH 8.2. Anadditional 10 ml of the ethanolamine solution was added, and the columnwas again capped and incubated overnight with rotation. The column bedwas washed with 20 ml of 1.5 M sodium chloride, 0.5 M Tris, pH 7.5,followed by a series of buffers containing 0.1 mM EDTA, 0.2% TritonX-100, and the following components; 20 mM sodium acetate, pH 4.5 (100ml); 20 mM sodium acetate, pH 4.5, 1.0 M sodium chloride (100 ml); 20 mMsodium borate, pH 9.5, 1.0 M sodium chloride (200 ml); 20 mM sodiumborate, pH 9.5 (100 ml). Finally, the column bed was washed with 15 mlof 2 mM Tris, 0.01% sodium azide (no Triton or EDTA), and stored in thatbuffer, at 4° C.

EXAMPLE 8 Co-Transfection of Cells with β-secretase and APP

293T cells were co-transfected with equivalent amounts plasmids encodingAPPsw or wt and β-secretase or control β-galalactoside (β-gal) cDNAusing FuGene 6 Reagent, as described in Example 4, above. EitherpCEKclone27 or pohCJ containing full length β-secretase were used forexpression of β-secretase. The plasmid construct pohCK751 used for theexpression of APP in these transfections was derived as described inDugan et al., JBC, 270(18) 10982–10989(1995) and shown schematically inFIG. 21. A β-gal control plasmid was added so that the total amount ofplasmid transfected was the same for each condition. β-gal expressingpCEK and pohCK vectors do not replicate in 293T or COS cells. Triplicatewells of cells were transfected with the plasmid, according to standardmethods described above, then incuabated for 48 hours, before collectionof conditioned media and cells. Whole cell lysates were prepared andtested for the β-secretase enzymatic activity. The amount of α-secretaseactivity expressed by transfected 293T cells was comparable to or higherthan that expressed by CosA2 cells used in the single transfectionstudies. Western blot assays were carried out on conditioned media andcell lysates, using the antibody 13G8, and Aβ ELISAs carried out on theconditioned media to analyze the various APP cleavage products.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All patent and literature references referred to herein areherein incorporated by reference.

1. A protein purified to apparent homogeneity comprising residues 63–452of SEQ ID NO:2, wherein the protein lacks amino acid residues 1–45 ofSEQ ID NO:2, and the protein exhibits β-secretase activity, and whereinthe protein is expressed from a bacterial source.
 2. The protein ofclaim 1, wherein said protein consists of SEQ ID NO:
 43. 3. The proteinof claim 1, wherein said protein has an N-terminal residue correspondingto a residue selected from the group consisting of residues 46, 58 and63 of SEQ ID NO: 2 and a C-terminus selected from a residue betweenpositions 452 and 501 of SEQ ID NO:2.
 4. The protein of claim 1, whereinsaid protein is produced by a heterologous cell.
 5. A crystallineprotein composition formed from the protein of claim
 1. 6. Thecrystalline protein composition of claim 5, wherein said purifiedprotein is characterized by a binding affinity for the β-secretaseinhibitor substrate P10-P4′sta D→V which is at least 1/100 of anaffinity exhibited by a protein having the amino acid sequence SEQ IDNO: 43, when said proteins are tested for binding to said substrateunder the same conditions.
 7. The crystalline protein composition ofclaim 5, wherein said composition is formed from a protein having asequence selected from the group consisting of SEQ ID NO: 43, and SEQ IDNO:
 71. 8. The crystalline protein composition of claim 5, wherein saidcomposition further includes a β-secretase substrate or inhibitormolecule.
 9. The crystalline protein composition of claim 8, whereinsaid β-secretase inhibitor consists of the sequence SEQ ID NO: 72[P10-P4′sta D→V].
 10. The crystalline protein composition of claim 8,wherein said β-secretase inhibitor consists of the sequence SEQ ID NO:81 [EVMXVAEF], wherein X is hydroxyethylene or statine.
 11. Thecrystalline protein composition of claim 8, wherein said β-secretaseinhibitor is characterized by a K_(i) of no more than about 50 μM. 12.The protein of claim 1, wherein the protein has been purifiedsufficiently to run as a single band on a SDS PAGE gel under reducingconditions.
 13. The protein of claim 1, wherein the protein has beenpurified sufficiently to provide a suitable substrate for N-terminalamino acid determination.
 14. The protein of claim 1, wherein theprotein comprises SEQ ID NO: 58.